Methods of diethylenetriaminepentaacetic acid (DTPA) quantification

Abstract

Provided herein are methods of determining the concentration of diethylenetriamene pentaacetate (DTPA) in a pharmaceutical formulation. In exemplary embodiments, the method comprises providing (i) a reference standard comprising, citrate and a known concentration of DTPA and (ii) a sample of a pharmaceutical formulation comprising DTPA, citrate and a protein; wherein the citrate concentration of the reference standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; wherein each of the reference standard and the sample of the pharmaceutical formulation further comprise iron and the iron concentration of the reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard and sample is in excess of the citrate present in each of the reference standard and sample, and applying each of the reference standard and sample to an RPLC column.

Description

10487-W001-SECMETHODS OF DIETHYLENETRIAMINEPENTAACETIC ACID (DTPA) QUANTIFICATIONCROSS REFERENCE TO RELATED APPLICATION

[0001] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 737,106, filed December 20, 2024, is hereby claimed.BACKGROUND

[0002] Chelating agents, such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA), are often included in pharmaceutical formulations, as their ability to chelate trace heavy metals inhibits or slows metal-mediated degradation of therapeutic proteins in pharmaceutical formulations, which ultimately elongates the shelf life of a pharmaceutical formulation. See, e.g., Wang and Tomasella, J Pharm Anal. 6(3):150-156 (2016).

[0003] With the inclusion of DTPA in pharmaceutical formulations, the need for accurate and precise analytical methods for reliable quantification of DTPA in pharmaceutical formulations arose. Quantification of DTPA is challenging, because DTPA does not contain a chromophore detectable by direct UV detection. Wang and Tomasella, 2016, supra, describes an ion-pairing HPLC method for determining the amount of DTPA in which DTPA is combined with iron to form a metallocomplex that is detectable by the method. As further discussed herein, the limitations of this HPLC method became apparent, however, when the method led to the repeated, inaccurate overestimation of the DTPA concentration in samples. Thus, methods that accurately and reliably quantify DTPA, e.g., without overestimating, are needed.SUMMARY

[0004] Provided herein for the first time is a new chromatographic method that efficiently and accurately quantifies DTPA in a pharmaceutical formulation. Without being bound to a scientific theory, it is postulated that the methods described herein are particularly useful for pharmaceutical formulations comprising DTPA and another excipient with metal-chelating activity, such as citrate.

[0005] Accordingly, provided herein are methods of determining the concentration of DTPA in a pharmaceutical formulation. In exemplary embodiments, the method comprises (a) applying each of a reference standard and a sample of a pharmaceutical formulation to a reversed-phase liquid chromatography (RPLC) column; wherein the reference standard comprises citrate and a known concentration of DTPA and the pharmaceutical formulation comprises DTPA, citrate, and a protein; wherein the citrate concentration of the reference10487-W001-SEC standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; wherein each of the reference standard and the sample of the pharmaceutical formulation further comprises iron, wherein the iron concentration of the reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard and sample is in excess of the citrate present in each of the reference standard and sample; (b) applying a mobile phase to the RPLC column to obtain chromatographic data for the sample and the reference standard; and (c) comparing the chromatographic data for the sample to the chromatographic data for the reference standard.

[0006] In exemplary embodiments, the method of determining the concentration of DTPA in a pharmaceutical formulation comprises (a)providing (i) a reference standard comprising citrate and a known concentration of DTPA and (ii) a sample of a pharmaceutical formulation comprising DTPA, citrate, and a protein; wherein the citrate concentration of the reference standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; wherein each of the reference standard and the sample of the pharmaceutical formulation further comprises iron, wherein the iron concentration of the reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard and sample is in excess of the citrate present in each of the reference standard and sample; (b) applying each of the reference standard and the sample of the pharmaceutical formulation to a reversed-phase liquid chromatography (RPLC) column; (c) applying a mobile phase to the RPLC column to obtain chromatographic data for the sample and the reference standard; and (d) comparing the chromatographic data for the sample to the chromatographic data for the reference standard. In various aspects, the citrate present in the reference standard is added and / or the citrate present in the sample of the pharmaceutical formulation is added. In various instances, the method further comprises adding citrate to the reference standard and / or the sample of the pharmaceutical formulation to provide the reference standard and / or the sample of the pharmaceutical formulation of (a). In exemplary aspects, the method comprises adding a citrate solution to the reference standard and / or the sample of the pharmaceutical formulation. In some aspects, the citrate concentration of the citrate solution is the same as the citrate concentration of the pharmaceutical formulation. In various aspects, the citrate concentration of the reference standard and / or the sample of the pharmaceutical formulation is the same as the citrate concentration of the pharmaceutical formulation. In various instances, the citrate concentration of the citrate solution, reference standard and / or the sample of the10487-W001-SEC pharmaceutical formulation is at least or about 15 mM, at least or about 20 mM, at least or about 50 mM. In various instances, the citrate concentration of the citrate solution, reference standard and / or the sample of the pharmaceutical formulation is less than or about 100 mM. The citrate in some aspects comprises sodium citrate. In various instances, the method comprises adding iron to the reference standard and / or the sample of the pharmaceutical formulation to provide the reference standard and / or the sample of the pharmaceutical formulation of (a). In exemplary aspects, the method comprises adding an iron solution to the reference standard and / or the sample of the pharmaceutical formulation. In exemplary instances, the iron comprises ferric chloride. The iron concentration of the iron solution is at least or about 0.0005% (w / v) or least or about 0.001% (w / v), in some aspects. In exemplary instances, the iron concentration of the iron solution is at least or about 0.005% (w / v), at least or about 0.010% (w / v), at least or about 0.025% (w / v), optionally, wherein the iron solution comprises about 0.005% (w / v) ferric chloride, about 0.010% (w / v) ferric chloride or about 0.025% (w / v) ferric chloride.

[0007] In exemplary embodiments, the method of determining the concentration of DTPA in a pharmaceutical formulation comprises (i) providing or preparing one or more reference standards, each comprising a known concentration of DTPA and citrate so that the citrate concentration of the reference standard(s) is the same as the citrate concentration of a sample of a pharmaceutical formulation and (ii) adding iron to each of the reference standard(s) and the sample of the pharmaceutical formulation so that the iron concentration of the reference standard(s) is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard(s) and the sample of the pharmaceutical formulation is in excess of the citrate. It will be appreciated that in some instances, a reference standard may already contain iron, at which point, adding iron to each of the reference standard(s) and the sample of the pharmaceutical formulation will be understood to include providing the reference standard that already contains iron, and adding iron to the sample of the pharmaceutical formulation.

[0008] In exemplary embodiments, the method comprises (a) adding a citrate solution to a series of reference standards, wherein each reference standard comprises a known DTPA concentration which is unique among the series of reference standards, whereupon the citrate concentration of each reference standard is the same as the citrate concentration of a sample of a pharmaceutical formulation comprising a protein, citrate and DTPA; (b) adding an iron solution to the sample and / or each reference standard of the series, whereupon (i) the iron concentration of each reference standard is the same as the iron concentration of the sample10487-W001-SEC of the pharmaceutical formulation and (ii) the iron present in each reference standard and the sample is in excess of the citrate present in each reference standard and the sample, (c) applying each reference standard and the sample to the RPLC column; (d) applying a mobile phase to the RPLC column to obtain chromatographic data for the sample and the series of reference standards; and (e) comparing the chromatographic data for the sample to the chromatographic data for the series of reference standards.

[0009] Additional exemplary embodiments and aspects of the presently disclosed methods are further described herein.BRIEF DESCRIPTION OFTHE DRAWINGS

[0010] Figure 1 A is an overlay of chromatograms obtained with two reference standards: one comprising EDTA and one comprising DTPA. A copper solution was added to each reference standard priorto chromatographic separation.

[0011] Figure 1 B is an overlay of chromatograms obtained with a DTPA reference standard and a pharmaceutical formulation sample comprising DTPA. An iron solution was added to each of the DTPA reference standard and the pharmaceutical formulation sample priorto chromatographic separation.

[0012] Figure 1 C is an overlay of chromatograms obtained with two DTPA reference standards, (DTPA Standard 1 having an expected DTPA concentration of 2.5 pM and DTPA Standard 5 having an expected DTPA concentration of 22.5 pM), and a pharmaceutical formulation sample. The overlay is an enlargement of the boxed peaks of the spectra shown in the upper right corner.

[0013] Figure 1 D is a graph of % DTPA spike recovery plotted as a function of spike level for the upperthreshold and lower threshold and forthe spiked samples.

[0014] Figure 2 is a graph of peak area plotted as a function of DTPA Standard Concentration for each of Sample Series A-D, as well as for positive and negative control series.

[0015] Figure 3 is a graph of peak area plotted as a function of DTPA Standard Concentration for each of Sample Series E-H.

[0016] Figure 4 is an overlay of chromatograms obtained with two DTPA reference standards, (DTPA Standard 1 and DTPA Standard 5) and a pharmaceutical formulation sample. The overlay is an enlargement of the boxed peaks of the spectra shown in the upper right corner.10487-W001-SEC

[0017] Figure 5 is a graph of the measured (recovered) DTPA concentration (as determined by the method of the present disclosure) plotted as a function of expected DTPA concentration.

[0018] Figure 6 comprises a pair of structures of DTPA complexed with Fe3+ (right) or without Fe3+ (uncomplexed; left) and an overlay of chromatograms obtained with two DTPA standards combined with or without ferric chloride prior to the chromatographic separation.DETAILED DESCRIPTION

[0019] Diethylenetriaminepentaacetic acid (DTPA), also known as pentetic acid, is an amino polycarboxylic acid having the formula C14H23N3O10 and CAS No. 67-43-6. Its IUPAC name is 2- [bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid. DTPA has the following structure:

[0020] Quantification of DTPA can be challenging, because DTPA does not contain a chromophore directly detectable with UV. While methods aimed at quantifying DTPA have been previously described, such methods, as demonstrated herein, do not reliably and accurately quantify DTPA in certain instances.

[0021] Described herein are methods that efficiently and accurately quantify DTPA in a pharmaceutical formulation. In various aspects, the presently disclosed methods are efficient and may be carried out in less than 90 minutes, less than 60 minutes, or less than 30 minutes. The methods of the present disclosure additionally do not require expensive and / or rare reagents. As described herein (see, e.g., Example 11 ), the methods of the present disclosure are advantageously highly sensitive, specific, precise, and repeatable. The methods of the present disclosure are highly accurate - the calculated accuracy was 100% and was based on R2of the measured vs. expected DTPA concentration being (0.9998, or 1 .00). The methods demonstrate a nearly perfect linearity or one-to-one correspondence between the expected or10487-W001-SEC calculated DTPA concentration and the measured or recovered DTPA concentration across a wide range of DTPA concentrations, e.g., about 0.0005 mM to about 0.0036 mM when the samples are diluted 10-fold, or about 0.005 mM to about 0.036 mM (when samples are not diluted). The methods are sensitive, exhibiting a sample limit of quantitation (LOQ) of about 0.2 pg / mL, for example. Also, the precision or repeatability of the presently disclosed methods are demonstrated by an overall % relative standard deviation (RSD) of 1 %. The overall recovery exhibited by the presently disclosed methods in spike recovery assays was 99%.

[0022] Accordingly, provided herein are methods of determining the concentration of DTPA in a pharmaceutical formulation. In exemplary embodiments, the method comprises (a) applying each of a reference standard and a sample of a pharmaceutical formulation to a reversed-phase liquid chromatography (RPLC) column; wherein the reference standard comprises citrate and a known concentration of DTPA and the pharmaceutical formulation comprises DTPA, citrate, and a protein; wherein the citrate concentration of the reference standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; wherein each of the reference standard and the sample of the pharmaceutical formulation further comprises iron, wherein the iron concentration of the reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard and sample is in excess of the citrate present in each of the reference standard and sample; (b) applying a mobile phase to the RPLC column to obtain chromatographic data for the sample and the reference standard; and (c) comparing the chromatographic data for the sample to the chromatographic data for the reference standard.

[0023] In exemplary embodiments, the method determining the concentration of DTPA in a pharmaceutical formulation comprises (a)providing (i) a reference standard comprising citrate and a known concentration of DTPA and (ii) a sample of a pharmaceutical formulation comprising DTPA, citrate, and a protein; wherein the citrate concentration of the reference standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; wherein each of the reference standard and the sample of the pharmaceutical formulation further comprises iron, wherein the iron concentration of the reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard and sample is in excess of the citrate present in each of the reference standard and sample; (b) applying each of the reference standard and the sample of the pharmaceutical formulation to a reversed-phase liquid chromatography (RPLC) column; (c) applying a mobile phase to the RPLC column to obtain chromatographic10487-W001-SEC data for the sample and the reference standard; and (d) comparing the chromatographic data for the sample to the chromatographic data for the reference standard. In various aspects, the citrate present in the reference standard is added and / or the citrate present in the sample of the pharmaceutical formulation is added. In various instances, the method further comprises adding citrate to the reference standard and / or the sample of the pharmaceutical formulation to provide the reference standard and / or the sample of the pharmaceutical formulation of (a). In exemplary aspects, the method comprises adding a citrate solution to the reference standard and / or the sample of the pharmaceutical formulation. In some aspects, the citrate concentration of the citrate solution is the same as the citrate concentration of the pharmaceutical formulation, which in various aspects, is the same as the citrate concentration of the reference standard and / or the sample of the pharmaceutical formulation. In various instances, the citrate concentration of the citrate solution, reference standard and / or the sample of the pharmaceutical formulation is at least or about 15 mM, at least or about 20 mM, at least or about 50 mM. In various instances, the citrate concentration of the citrate solution, reference standard and / or the sample of the pharmaceutical formulation is less than or about 100 mM. The citrate in some aspects comprises sodium citrate. In various instances, the method comprises adding iron to the reference standard and / or the sample of the pharmaceutical formulation to provide the reference standard and / or the sample of the pharmaceutical formulation of (a). In exemplary aspects, the method comprises adding an iron solution to the ref’aerence standard and / or the sample of the pharmaceutical formulation. In exemplary instances, the iron comprises ferric chloride. The iron concentration of the iron solution is at least or about 0.0005% (w / v) or least or about 0.001 % (w / v), in some aspects. In exemplary instances, the iron concentration of the iron solution is at least or about 0.005% (w / v), at least or about 0.010% (w / v), at least or about 0.025% (w / v), optionally, wherein the iron solution comprises about 0.005% (w / v) ferric chloride, about 0.010% (w / v) ferric chloride or about 0.025% (w / v) ferric chloride.

[0024] In exemplary embodiments, the method of determining the concentration of DTPA in a pharmaceutical formulation comprises (i) providing or preparing one or more reference standards, each comprising a known concentration of DTPA and citrate so that the citrate concentration of the reference standard(s) is the same as the citrate concentration of a sample of a pharmaceutical formulation and (ii) adding iron to each of the reference standard(s) and the sample of the pharmaceutical formulation so that the iron concentration of the reference standard(s) is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard(s) and the10487-W001-SEC sample of the pharmaceutical formulation is in excess of the citrate. It will be appreciated that in some instances, a reference standard may already contain iron, at which point, adding iron to each of the reference standard(s) and the sample of the pharmaceutical formulation will be understood to include providing the reference standard that already contains iron, and adding iron to the sample of the pharmaceutical formulation. It will be appreciated that in some instances, the sample of the pharmaceutical formulation may already contain iron, at which point, adding iron to each of the reference standard(s) and the sample of the pharmaceutical formulation will be understood to include providing the sample of the pharmaceutical formulation that already contains iron, and adding iron to reference standard(s). When it is said that the iron concentration of the reference standard(s) is the “same” as the iron concentration of the sample, it will be appreciated that negligible variation, such as experimental error orthe presence of residual trace amounts of iron in the sample (or reference standard) in addition to the iron that has been added, will remain within this scope. For example, in some instances, the sample of the pharmaceutical formulation comprises a trace amount of iron such that the amount of iron added to the sample upon the method of the present invention is considered the same as the amount of iron added to the reference standard(s), since the added amount of iron is determinative of the performance of the sample in the method, regardless of the presence or absence of the trace amount. In various aspects, the pharmaceutical formulation being assayed for DTPA concentration lacks iron or comprises a negligible amount of iron, e.g., an amount of iron which is within acceptable limits of a metal leachable, so that the amount of iron added to the sample of the pharmaceutical formulation is considered the same as the amount of iron added to the reference standard.

[0025] In exemplary embodiments, the method of determining the concentration of DTPA in a pharmaceutical formulation comprises adding citrate to one or more reference standards to match the citrate concentration of a sample of a pharmaceutical formulation and / or adding iron to each of the reference standard(s) to match the iron concentration of the sample of the pharmaceutical formulation. In various aspects, the method comprises adding a citrate solution and / or an iron solution. In exemplary aspects, the citrate (e.g., citrate solution) is added to each of the reference standard(s) and the sample and / or the iron (e.g., iron solution) is added to each of the reference standard(s) and the sample, whereupon the citrate concentration and the iron concentration of each of the reference standard(s) are the same as the citrate concentration and the iron concentration of the sample of the pharmaceutical formulation.10487-W001-SEC

[0026] In exemplary embodiments, the method of determining a concentration of DTPA in a pharmaceutical formulation comprises (a) adding citrate to a reference standard comprising a known concentration of DTPA, whereupon the citrate concentration of the reference standard is the same (e.g., ±10%) as the citrate concentration of a sample of a pharmaceutical formulation comprising DTPA, citrate and a protein, (b) adding iron to each of the reference standard and the sample of the pharmaceutical formulation, whereupon (i) the iron concentration of the reference standard is the same (e.g., ±10%) as the iron concentration of the sample of the pharmaceutical formulation and (ii) the iron present in each of the reference standard and sample is in excess of the citrate present in each of the reference standard and sample; (c) applying each of the sample and the reference standard to a reversed phase liquid chromatography (RPLC) column; (d) applying a mobile phase to the RPLC column to obtain chromatographic data forthe sample and the reference standard, and (e) comparing the chromatographic data forthe sample to the chromatographic data forthe reference standard.

[0027] As used herein, the term “reference standard” refers to a sample comprising a known amount of DTPA. In some aspects, (for example, when setting up a curve), the reference standard may also be accompanied by a negative control or “blank” in which DTPA is known to be absent. In various aspects, the reference standard consists or consists essentially of DTPA. The reference standard in exemplary aspects, comprises a concentration of DTPA within a given DTPA concentration range. The DTPA concentration range in various aspects is about 0.00025 mM to about 1 mM, about 0.00025 mM to about 0.1 mM, about 0.00025 mM to about 0.01 mM, about 0.00025 mM to about 0.001 mM, about 0.00025 mM to about 0.0025 mM, about 0.00025 mM to about 0.025 mM, about 0.00025 mM to about 0.25 mM, about 0.00025 mM to about 2.5 mM, about 0.0025 mM to about 2.5 mM, about 0.025 mM to about 2.5 mM, 0.25 mM to about 2.5 mM, about 0.001 mM to about 1 mM, about 0.01 mM to about 1 mM, or about 0.1 mM to about 1 mM. In various aspects, the DTPA concentration range is about 0.0005 mM to about 0.0036 mM or about 0.0005 mM to about 0.0050 mM. In exemplary embodiments, the reference standard is one of a series of reference standards, each reference standard of the series having a DTPA concentration different from another reference standard of the series. In exemplary embodiments, the method comprises (a) adding citrate, e.g., a citrate solution, to a series of reference standards, wherein each reference standard comprises a known DTPA concentration which is unique among the series of reference standards, whereupon the citrate concentration of each reference standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; (b) adding iron, e.g., an iron solution, to each reference standard of the series, whereupon (i) the iron concentration of10487-W001-SEC each reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and (ii) the iron present in each reference standard and the sample is in excess of the citrate; (c) applying each reference standard and the sample to an RPLC column; (d) applying a mobile phase to the RPLC column to obtain chromatographic data forthe sample and the series of reference standards; and (e) comparing the chromatographic data forthe sample to the chromatographic data forthe series of reference standards. In exemplary aspects, the series comprises a reference standard comprising a minimum DTPA concentration and a reference standard comprising a maximum DTPA concentration, wherein the maximum DTPA concentration is at least 2 times the minimum DTPA concentration. In various aspects, the maximum DTPA concentration is at least 5 times the minimum DTPA concentration. In various instances, the maximum DTPA concentration is at least 10 times the minimum DTPA concentration. The minimum DTPA concentration and maximum DTPA concentration in various aspects are about 0.00025 mM and about 1 mM, respectively, about 0.00025 mM and about 0.1 mM respectively, about 0.00025 mM and about 0.01 mM, respectively, about 0.00025 mM and about 0.001 mM, respectively, about 0.00025 mM and about 0.0025 mM, respectively, about 0.00025 mM and about 0.025 mM, respectively, about 0.00025 mM and about 0.25 mM, respectively, about 0.00025 mM and about 2.5 mM, respectively, about 0.0025 mM and about 2.5 mM, respectively, about 0.025 mM and about 2.5 mM, respectively, 0.25 mM and about 2.5 mM, respectively, about 0.001 mM and about 1 mM, respectively, about 0.01 mM and about 1 mM, respectively, or about 0.1 mM and about 1 mM, respectively. In various aspects, the minimum DTPA concentration and maximum DTPA concentration are about 0.0005 mM and about 0.0036 mM, respectively, or about 0.0005 mM and about 0.0050 mM, respectively. In exemplary aspects, the minimum DTPA concentration and maximum are about 0.0005 mM and about 0.0036 mM, respectively.

[0028] In exemplary aspects, the pharmaceutical formulation comprises DTPA, citrate, and a protein (e.g., a therapeutic protein). In exemplary instances, the pharmaceutical formulation comprises an unknown DTPA concentration or an estimated DTPA concentration, and the method determines or confirms the DTPA concentration of the pharmaceutical formulation. In various instances, the pharmaceutical formulation further comprises citrate and a protein (e.g., a therapeutic protein). The presently disclosed methods are advantageously not limited to the type, structure, or identity of the protein, and thus the protein may be any protein known in the art, for example a therapeutic protein such as a canonical monoclonal antibody, antigen binding protein (e.g., a bispecific ortrispecific antibody), fusion protein, or mutein). In exemplary instances, the pharmaceutical formulation comprises a known amount of citrate.10487-W001-SECIn exemplary aspects, the pharmaceutical formulation comprises at least or about 1 mM citrate, at least or about 5 mM citrate or at least or about 10 mM citrate. In various instances, the pharmaceutical formulation comprises at least or about 15 mM citrate. In various instances, the pharmaceutical formulation comprises at least or about 20 mM citrate. In various instances, the pharmaceutical formulation comprises at least or about 30 mM citrate. In various instances, the pharmaceutical formulation comprises at least or about 40 mM citrate. In various instances, the pharmaceutical formulation comprises at least or about 50 mM citrate. In exemplary aspects, the pharmaceutical formulation comprises less than about 200 mM citrate. In exemplary aspects, the pharmaceutical formulation comprises less than about 150 mM citrate. In exemplary aspects, the pharmaceutical formulation comprises less than about 100 mM citrate. In various instances, the pharmaceutical formulation comprises citrate in a range between two of the listed values, for example 1 mM - 100 mM citrate, 1 mM - 50 mM citrate, 5 mM - 100 mM citrate, 5 mM - 50 mM citrate, 10 mM - 100 mM citrate, or 10 mM - 50 mM citrate. The presently disclosed methods are advantageously not limited to the type of citrate in the pharmaceutical formulation and thus the citrate can be any type of citrate. In various aspects, the citrate is sodium citrate. In various aspects, the pharmaceutical formulation comprises a drug substance (DS) or drug product (DP) or an in- process sample, e.g., a drug substance produced following an ultrafiltration / diafiltration (UF / DF). In various aspects, the citrate is a component of a pharmaceutical formulation. In exemplary aspects, the sample of the pharmaceutical formulation is a dilution of the pharmaceutical formulation, optionally, wherein the citrate concentration of the sample is the same as the citrate concentration of the pharmaceutical formulation. In exemplary instances, the sample of the pharmaceutical formulation is an undiluted aliquot of the pharmaceutical formulation and the citrate concentration of the sample is the same as the citrate concentration of the pharmaceutical formulation.

[0029] In exemplary aspects, the methods of the present disclosure comprise preparing or providing one or more reference standards with citrate so that the citrate concentration of the reference standard(s) is the same as the citrate concentration of the sample of the pharmaceutical formulation. In exemplary instances, the presently disclosed methods comprise providing a reference standard that already contains citrate. In exemplary instances, the presently disclosed methods comprise adding a citrate solution to a reference standard, whereupon the citrate concentration of the reference standard is the same (e.g., ±10%) as the citrate concentration of the sample of the pharmaceutical formulation. A person of ordinary skill in the art will appreciate that small amounts of variation (due to, e.g.,10487-W001-SEC experimental error or differences in instrument sensitivity) is permitted between two compositions considered to have “the same” concentration or amount of an ingredient (such as, e.g., citrate or iron). That is to say, a trivial difference in citrate and / or iron concentration may exist between the reference standard and a sample of the pharmaceutical formulation, but the concentrations will be considered to be “the same” for the purposes of the methods hereon. If further numerical precision is required, it will be appreciated that an amount of citrate or iron between a reference standard and a pharmaceutical formulation (or vice versa, and including applicable dilutions of these compositions) will be considered “the same” when a first value is within a range that is 10% less and 10% greater than a second value. In various aspects, the citrate concentration of the reference standard is within a range that is 10% less and 10% greater than the citrate concentration of the sample of the pharmaceutical formulation. Citrate may be added to the reference standard and / or pharmaceutical formulation in a solution. In exemplary aspects, the citrate solution used in the method of the present disclosure is a solution comprising at least or about 1 mM citrate, at least or about 5 mM citrate or at least or about 10 mM citrate. In various instances, the citrate solution comprises at least or about 15 mM citrate. In various instances, the citrate solution comprises at least or about 20 mM citrate. In various instances, the citrate solution comprises at least or about 30 mM citrate. In various instances, the citrate solution comprises at least or about 40 mM citrate. In various instances, the citrate solution comprises at least or about 50 mM citrate. In exemplary aspects, the citrate solution comprises less than about 200 mM citrate. In exemplary aspects, the citrate solution comprises less than about 150 mM citrate. In exemplary aspects, the citrate solution comprises less than about 100 mM citrate. In various aspects, the citrate solution comprises a citrate concentration that is the same (e.g., ±10%) as the citrate concentration of the pharmaceutical formulation. Optionally, the citrate concentration of the citrate solution is about 10 mM to about 50 mM, about 15 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, about 35 mM to about 50 mM, about 40 mM to about 50 mM, about 45 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, or about 10 mM to about 15 mM. In various aspects, the citrate solution comprises about 20 mM citrate. In various aspects, the citrate, e.g., citrate solution, comprises sodium citrate. In various instances, the citrate, e.g., citrate solution, comprises trisodium citrate dihydrate, monosodium citrate, disodium citrate, disodium citrate sesquihydrate, anhydrous trisodium citrate, and the like. In various aspects, the citrate, e.g., citrate solution, comprises another10487-W001-SEC source of citrate, e.g., triethyl citrate, potassium citrate, monostea ryl citrate, glyceryl monocitrate, disodium hydrogen citrate, calcium citrate, acetyl butyl citrate, citric acid monohydrate. In various aspects, the citrate, e.g., citrate solution, comprises sodium citrate. Optionally, citrate is added in a solution, and the citrate solution comprises about 10 mM to about 50 mM, about 15 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, about 35 mM to about 50 mM, about 40 mM to about 50 mM, about 45 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, or about 10 mM to about 15 mM sodium citrate. Optionally, the citrate solution is 20 mM sodium citrate.

[0030] It will be appreciated that citrate may be provided as a pharmaceutically acceptable salt. By way of example, citrate may be provided as trisodium citrate dihydrate, triethyl citrate, potassium citrate, monostearyl citrate, monosodium citrate, glyceryl monocitrate, disodium hydrogen citrate, disodium citrate sesqui hydrate, calcium citrate, anhydrous trisodium citrate, acetyltributyl citrate, or citric acid monohydrate.

[0031] In exemplary aspects, the methods of the present disclosure comprise preparing one or more reference standards and the sample of the pharmaceutical formulation with citrate so that the citrate concentration of the reference standard(s) is the same as the citrate concentration of the sample of the pharmaceutical formulation. In exemplary instances, the presently disclosed methods comprise adding a citrate (for example, as a citrate solution) to both the reference standard and the sample of the pharmaceutical formulation, whereupon the citrate concentration of the reference standard is the same (e.g., ±10%) as the citrate concentration of the sample of the pharmaceutical formulation. In various aspects, adding the citrate to the sample of the pharmaceutical formulation dilutes the sample. Optionally, adding the citrate to the sample dilutes the sample of the pharmaceutical formulation about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or more.

[0032] In exemplary aspects of the present disclosure, the methods comprise adding an iron (for example, as an iron solution solution) to each of the reference standard(s) and the sample of the pharmaceutical formulation so that the iron concentration of the reference standard(s) is the same as the iron concentration of the sample of the pharmaceutical formulation and the iron present in each of the reference standard(s) and the sample of the pharmaceutical formulation is in excess of the citrate. As used herein, the phrase “in excess of” refers to that the amount of one component, e.g., iron, being greaterthan the amount of another component, e.g., citrate. In various aspects, the iron present in each of the reference10487-W001-SEC standard(s) and the sample of the pharmaceutical formulation is in excess of the citrate means that the amount of iron in the reference standard or sample of the pharmaceutical formulation is greaterthan the amount of citrate in that reference standard or sample. Without being limited by theory, sufficient iron should be present so that DTPA is bound by iron, and is optically detectable in the chromatographic analysis. It will be appreciated that an excess of iron is present when incremental increases in the amount of iron do not affect the area under the peak for DTPA in a chromatogram. In exemplary instances, the amount of iron (w / v) % is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% greaterthan the amount of citrate. In various aspects, after adding the iron, e.g., as an iron solution, the iron present in the reference standard(s) or sample of the pharmaceutical formulation is at least 1 .5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 10-fold, at least 15- fold, at least 20-fold the citrate present in the reference standard or sample of the pharmaceutical formulation. For each reference standard and sample of the pharmaceutical formulation used in the presently disclosed method, the iron is in excess of the citrate, after adding the iron. The iron may be added in an iron solution. In exemplary aspects, the iron concentration of the iron solution is at least 0.001 % (w / v) or at least 0.005% (w / v). In various aspects, the iron concentration of the iron solution is at least 0.025% (w / v). In various aspects, the iron solution comprises Fe2+or Fe3+. In exemplary aspects, the iron solution comprises ferrous chloride. In various instances, the iron solution is 0.005% (w / v) ferrous chloride, 0.010% (w / v) ferrous chloride, or 0.025% (w / v) ferrous chloride. In exemplary aspects, the iron solution ferric chloride. In various instances, the iron solution is 0.005% (w / v) ferric chloride, 0.010% (w / v) ferric chloride, or 0.025% (w / v) ferric chloride. In exemplary aspects, the iron solution comprises ferric phosphate or ferric oxide. In various aspects, the iron concentration of the reference standard is within a range that is 10% less and 10% greaterthan the iron concentration of the sample of the pharmaceutical formulation.

[0033] In exemplary embodiments, the presently disclosed methods comprise applying each of the sample of the pharmaceutical formulation and the reference standard(s) to a reversed phase liquid chromatography (RPLC) column and applying a mobile phase to the RPLC column to obtain chromatographic data for the sample of the pharmaceutical formulation and the reference standard. In various aspects, the method comprises RPLC which is a chromatographic separation technique used to separate components of the solutions applied to the RPLC column. As used herein, “separating” or “separation” refers to chromatographically separating or chromatographic separation or separation achieved10487-W001-SEC through chromatography, a technique in which the components of a mixture are separated by their distribution between two phases, a stationary phase and a mobile phase, based on the strength of the physical interactions, e.g., intermolecular interactions, made between the components of the mixture and the two phases. The separation results from repeated sorption / desorption events that occur during the movement of the mixture along the stationary phase in the general direction of the migration of the mobile phase applied to the stationary phase. See, e.g., Poole, “Chromatography”. Encyclopedia of Separation Science, pgs. 40-64, Elsevier Science, 2000. Accordingly, in exemplary embodiments, the methods of the present invention comprise chromatographic separation. In various embodiments, the methods of the present disclosure employ reversed phase liquid chromatography (RPLC) in which a non-polar stationary phase and a polar mobile phase are used to separate molecules based on their hydrophobicity. In various aspects, the presently disclosed methods employ reversed phase high-performance liquid chromatography (RP-HPLC) wherein the less hydrophobic molecules which bind to the non-polar stationary phase elute earlier than the more hydrophobic molecules. The theory behind RP-HPLC is detailed in Josie and Kovac, Curr Protoc Protein Sci 61 (1): 8.7.1 -8.7.22 (2010), https: / / doi.Org / 10.1002 / 0471140864. ps0807s61 ; and Kumar and Kumar, Int J Pharm Sci Res 3(12): 4626-4633 (2012). In various aspects, the RPLC column comprises a non-polar stationary phase. The non-polar stationary phase comprises hydrocarbons, e.g., C1 hydrocarbons, C4 hydrocarbons, C5 hydrocarbons, C8 hydrocarbons, C18 hydrocarbons, and the like, in various aspects. In various instances, the non-polar stationary phase comprises C18 hydrocarbons. In exemplary aspects, the hydrocarbons are linked or bound to the surface of particles of the non-polar stationary phase and the extent of the surface comprising linked hydrocarbons can vary. In various instances, at least about 15% of the surface of the particles of the non-polar stationary phase are linked to a hydrocarbon, e.g., C18 hydrocarbon. In various instances, at least about 25% or at least about 50% or at least about 75% of the surface of the particles of the non-polar stationary phase are linked to a hydrocarbon, e.g., C18 hydrocarbon. In exemplary embodiments, the non-polar stationary phase comprises particles comprising a silica, a hybrid silica, such as, for instance, a polyorganoethoxysilane. In exemplary instances, the particles comprise tetraethoxysilane (TEOS) and bis(triethoxysilyl) ethane (BTEE). In exemplary instances, the particles are any of those described in U.S. Patent Nos. 4,017,528; 6,686,035; 7,723,473; and 7,250,214. In various aspects, the non-polar stationary phase comprises ethylene-bridged-hybrid (BEH) particles. The RPLC column comprises a non-polar stationary phase comprising a surface charge, in various aspects. In some aspects, the RPLC column comprises a non-polar10487-W001-SEC stationary phase comprising charged surface hybrid (CSH) particles. In various aspects, the particles of the non-polar stationary phase have a particle size of about 1 pm to about 50 pm, about l pm to about 40 pm, about 1 pm to about 30 pm, about 1 pm to about 20 pm, about 1 pm to about 10 pm, or about 1 pm to about 5 pm. In various aspects, the particles of the nonpolar stationary phase have a particle size of about 5 pm. The particles of the non-polar stationary phase in exemplary aspects have a pore size of about 80 A to about 120 A or about 120 A to about 300 A. In various instances, the non-polar stationary phase comprises particles comprising a pore size of about 120 A to about 160 A. RPLC columns are commercially available through vendors, e.g., Waters (Milford, MA), Tosoh (Portland, OR), ThermoFisher Scientific (Waltham, MA), Agilent (Santa Clara, CA), Pheomenex (Torrance, CA), Hamilton Co. (Reno, NV), Restek (Center County, PA), EMD Millipore (St. Louis, MO), BioRad (Hercules, CA), Hawach Scientific (Xi’an City, PR China), Macherey-Nagel (Duren, Nordrhein-Westfalen, Germany), Halo Columns (Wilmington, DE), Sigma-Aldrich (St. Louis, MO), Develosil (San Diego, CA), Sartorius (Gottingen, Germany), Concise Separations (San Jose, CA), among others.

[0034] In exemplary embodiments of the presently disclosed methods, a mobile phase is applied to the RPLC column to elute bound components of the sample or reference standard(s) from the column to obtain chromatographic data for each of the sample of the pharmaceutical formulation and the reference standard(s). In exemplary aspects, the mobile phase comprises an iron pairing agent. The ion pairing agent is in exemplary instances tetrabutylammonium hydroxide (TBAH). In various aspects, the mobile phase comprises about 10 mM to about 50 mM ion pairing agent. In various aspects, the mobile phase comprises about 10 mM to about 40 mM ion pairing agent, about 10 mM to about 30 mM ion pairing agent, about 10 mM to about 20 mM ion pairing agent, about 10 mM to about 50 mM ion pairing agent. In various aspects, the mobile phase comprises about 20 mM to about 30 mM ion pairing agent, optionally about 22 mM to about 26 mM ion pairing agent, e.g., about 24 mM ion pairing agent. In exemplary aspects, the mobile phase further comprises an acid, such as phosphoric acid, for instance. In various instances, the mobile phase comprises about 0.05% (v / v) to about 0.50% (v / v) acid, e.g., phosphoric acid, optionally, about 0.15% (v / v) acid, e.g., phosphoric acid. In various aspects, the mobile phase is applied to the column for less than 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, or about 5 minutes. In exemplary instances, the method further comprises a column cleaning after applying the mobile phase. The column cleaning is carried out with an acetonitrile solution, in various aspects. In exemplary aspects, the method further10487-W001-SEC comprises applying an acetonitrile solution to the column after applying the mobile phase. In exemplary instances, the acetonitrile solution is about 5% (v / v) to about 50% (v / v) acetonitrile, optionally, about 20% (v / v) to about 30% (v / v) acetonitrile, e.g., about 25% (v / v) acetonitrile. In exemplary instances, the acetonitrile solution is applied to the column for less than 10 minutes, optionally, about 5 minutes. In various aspects, the method further comprises a column equilibration after applying the mobile phase. The column equilibration is carried out with the same mobile phase solution previously applied to the column. In various aspects, the mobile phase is applied in less than about 30 minutes, less than about 20 minutes, less than about 10 minutes, e.g., about 5 minutes. In various aspects, the method comprises applying the mobile phase to the RPLC for about 5 minutes, followed by a column cleaning and a column equilibration, each of which is 5 minutes, for a total of about 15 minutes.

[0035] In exemplary aspects, the method of the present disclosure is carried out with a flow rate of the reference standard or sample and mobile phase through the RPLC column of about 0.1 mL / minute to about 20 mL / minute, about 0.1 mL / minute to about 15 mL / minute, about 0.1 mL / minute to about 10 mL / minute, about 0.1 mL / minute to about 5 mL / minute, about 0.1 mL / minute to about 2.5 mL / minute, or about 0.1 mL / minute to about 1 mL / minute. In exemplary aspects, the flow rate is less than 1 mL per minute, e.g., about 0.5 mL / minute. In exemplary instances, the column length of the RPLC column is less than about 200 mm, less than about 100 mm, less than about 75 mm. In some instances, the column length of the RPLC column is about 50 mm. In various instances, the inner diameter of the RPLC column is about 1 mm to about 9 mm, e.g., about 1 mm to about 8 mm, about 1 mm to about 7 mm, about 1 mm to about 6 mm, about 1 mm to about 5 mm, about 1 mm to about 4 mm, about 1 mm to about 3 mm, about 1 mm to about 2 mm, about 2 mm to about 9 mm, about 3 mm to about 9 mm, about 4 mm to about 9 mm, about 5 mm to about 9 mm, about 6 mm to about 9 mm, about 7 mm to about 9 mm, or about 8 mm to about 9 mm. In exemplary aspects, the inner diameter of the RPLC column is about 4.6 mm. In various aspects, the injection volume is about l uL to about 50 uL, about 1 uL to about 25 uL, about 1 uL to about 15 uL, about 1 uL to about 10 uL, about 1 uL to about 5 uL. In various aspects, the injection volume is about 10 pL. In some instances, the temperature of the RPLC column is greater than 30 degrees C or greater than 35 degrees C. In various instances, the temperature of the RPLC column is about 40 degrees C. In various aspects, the RPLC column is connected to a system that comprises a detector, optionally, a diode array detector. The detector in various aspects detects at a detection wavelength greater than 260 nm, optionally, greater than 300 nm. In various instances, the detector detects at a detection wavelength of about 335 nm.10487-W001-SEC

[0036] During the presently disclosed method, e.g., while the mobile phase is applied, or shortly thereafter, chromatographic data is obtained. In various aspects, the chromatographic data comprises the peak area of one or more peaks of the chromatogram(s) for the sample(s) of the pharmaceutical formulation or the reference standard(s). In exemplary aspects, the chromatographic data for the sample of the pharmaceutical formulation comprise peak area of one or more peaks of the chromatogram of the sample. In exemplary aspects, the chromatographic data for the reference standard(s) comprise peak area of one or more peaks of the chromatogram(s) of the reference standard(s). In exemplary embodiments, the presently disclosed methods comprise comparing the chromatographic data for the sample to the chromatographic data for the reference standard. In various aspects, the method comprises comparing the peak area of a peak of the sample of the pharmaceutical formulation to the peak area of a corresponding peak of the reference standard.

[0037] In various aspects, the chromatographic data comprises peak area and the method comprises creating a standard curve, e.g., a linear regression curve, which correlates the peak area of the peak of each reference standard to the expected or known concentration of DTPA of the reference standard. In exemplary aspects, a linear equation for the standard curve is determined which linear equation allows input of the peak area of the sample of the pharmaceutical formulation to determine the DTPA concentration of the sample of the pharmaceutical formulation (e.g., a measured DTPA concentration).

[0038] Proteins

[0039] In exemplary embodiments, the protein is a therapeutic protein. As used herein, the term “therapeutic protein” refers to any molecule, which may be naturally-occurring or engineered or synthetic, comprising at least one polypeptide chain, which, when administered to a subject, is intended for achieving a therapeutic effect for treatment of a disease or medical condition. Therapeutic proteins can include antigen binding proteins such as antibodies and proteins comprising antibody fragments, as well as growth factors and enzymes. In exemplary instances, the protein, e.g., therapeutic protein, comprises one or more domains of an antibody, e.g., antibody domains, immunoglobulin domains. As used herein, the term “antibody” which is synonymous with “immunoglobulin”, refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions, as described in, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 4thed., Elsevier Science Ltd. / Garland Publishing, 1999. For example, an antibody may be an IgG which is a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about10487-W001-SEC25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In IgG formats, the variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and varies among other antibodies that bind to different antigens. The constant region of the antibody functions to recruit cells and molecules of the immune system. The variable region is made of the N- terminal regions of each light chain and heavy chain, while the constant region is made of the C-terminal portions of each of the heavy and light chains. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4thed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0040] The general structure and properties of CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition. A variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901 -917;Chothia et al., 1989, Nature 342: 877-883), within a framework region (designated framework regions 1 -4, FR1 , FR2, FR3, and FR4, by Kabat et al., 1991 ; see also Chothia and Lesk, 1987, supra).

[0041] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype is frequently utilized for therapeutic proteins. IgG has several subclasses, including, but not limited to IgG 1 , lgG2, lgG3, and lgG4. IgM has subclasses, including, but not limited to, IgM 1 and lgM2. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In exemplary embodiments, the antibody is an antibody of isotype IgG, including any one of IgG 1 , lgG2, lgG3 or lgG4. In exemplary aspects, the therapeutic protein comprises one or more antibody heavy chains.

[0042] An antibody can be cleaved into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab’)2fragment and a pFc’ fragment. In exemplary10487-W001-SEC aspects of the present disclosure, the fusion protein of the present disclosure comprises an antigen binding antibody fragment. As used herein, the term “antigen binding antibody fragment” or “antigen-binding fragment” or “antigen-binding portion” refers to a portion of an antibody that is capable of bindingto the antigen of the antibody. In exemplary instances, the antigen binding antibody fragment comprises a Fab fragment or a F(ab’)2fragment. Optionally, the protein of which the digested peptides are separated comprises an Fc domain and / or a Fab fragment.

[0043] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12-150 kDa and has a valency (n) range from monomeric (n = 1 ), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher; such alternative formats are referred to herein as “antibody protein products”. Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs and VHH / VH (discussed below). The smallest antigen binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble, flexible amino acid peptide linker is used to connect the V regions to a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. The smallest fragments are VHH / VH of camelid heavy chain Abs as well as single domain Abs (sdAb). The building block that is most frequently used to create novel antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which comprises V domains from the heavy and light chain (VH and VL domain) linked by a peptide linker of ~15 amino acid residues. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are well-described in the art. See, e.g., Shimamoto et al., mAbs 4(5): 586-591 (2012). Other antibody protein products include a single chain antibody (SCA); a diabody; a triabody; a tetrabody; bispecific or trispecific antibodies, and the like. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015). In various instances, the protein, e.g., therapeutic10487-W001-SEC protein, of which the digested peptides are se is an antibody or comprises a single chain variable (scFv) domain or is a bispecific antibody.

[0044] In exemplary embodiments, the therapeutic protein comprises an antibody heavy chain variable region and / or an antibody light chain variable region. The therapeutic protein is an antibody in various aspects. In exemplary instances, the therapeutic protein is an antigenbinding antibody fragment, for example, an scFv or scFab. In various aspects, the protein is a protein of an in-process sample, a drug substance, or drug product. In exemplary instances, the therapeutic protein is any one of the therapeutic proteins described herein at “Therapeutic Proteins”.

[0045] Therapeutic Proteins

[0046] The following disclosure is provided merely to illustrate therapeutic proteins of the present invention and not in any way to limit its scope. The therapeutic protein may be any of the following therapeutic proteins: CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34; including those that interfere with receptor binding. HER receptor family proteins, including HER2, HER3, HER4, and the EGF receptor. Cell adhesion molecules, for example, LFA-I, Mol, pl50, 95, VLA-4, ICAM-I, VCAM, and alpha v / beta 3 integrin. Growth factors, such as vascular endothelial growth factor (“VEGF”), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-I -alpha), erythropoietin (EPO), nerve growth factor, such as NGF- beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGF- a and TGF-p, including TGF-pi, TGF-02, TGF-03, TGF- (34, or TGF- 05, insulin-like growth factors-l and -II (IGF-I and IGF-II), des(l-3)-IGF-l (brain IGF-I), and osteoinductive factors. Insulins and insulin-related proteins, including insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins. Coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrands factor, protein C, alpha-1 -antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens. Colony stimulating factors and receptors thereof, including the following, among others, M-CSF, GM- CSF, and G-CSF, and receptors thereof, such as CSF-1 receptor (c-fms). Receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors (“TPO-R,” “c-mpl”),10487-W001-SEC glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c-Kit, and other receptors. Receptor ligands, including, for example, OX40L, the ligand for the 0X40 receptor. Neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A-chain, relaxin B-chain, and prorelaxin; interferons and interferon receptors, includingfor example, interferon-a, -0, and -y, and their receptors. Interleukins and interleukin receptors, including IL-I to IL-33 and IL-I to IL-33 receptors, such as the IL-8 receptor, among others. Viral antigens, including an AIDS envelope viral antigen. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, and activin. Integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies. Myostatins, TALL proteins, includingTALL-l, amyloid proteins, including but not limited to amyloid-beta proteins, thymic stromal lymphopoietins (“TSLP”), RANK ligand (“RANKL” or “OPGL”), c-kit, TNF receptors, includingTNF ReceptorType 1 , TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing. The therapeutic protein in some aspects, is a protein which binds to any one of the aforementioned proteins.

[0047] Additional exemplary therapeutic proteins include Activase® (Alteplase); alirocumab, Aranesp® (Darbepoetin-alfa), Epogen® (Epoetin alfa, or erythropoietin); Avonex® (Interferon p- la); Bexxar® (Tositumomab); Betaseron® (Interferon-P); bococizumab (anti-PCSK9 monoclonal antibody designated as L1 L3, see US8080243); Campath® (Alemtuzumab); Dynepo® (Epoetin delta); Velcade® (bortezomib); MLN0002 (anti-a4p7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); Enbrel® (etanercept); Eprex® (Epoetin alfa); Erbitux® (Cetuximab); evolocumab; Genotropin® (Somatropin); Herceptin® (Trastuzumab); Humatrope® (somatropin [rDNA origin] for injection); Humira® (Adalimumab); Infergen® (Interferon Alfacon-1 ); Natrecor® (nesiritide); Kineret® (Anakinra), Leukine® (Sargamostim); LymphoCide® (Epratuzumab); BenlystaTM (Belimumab); Metalyse® (Tenecteplase); Mircera® (methoxy polyethylene glycol-epoetin beta); Mylotarg® (Gemtuzumab ozogamicin); Raptiva® (efalizumab); Cimzia® (certolizumab pegol); SolirisTM (Eculizumab); Pexelizumab (Anti-C5 Complement); MEDI-524 (Numax®); Lucentis® (Ranibizumab); Edrecolomab (.Panorex®); Trabio® (lerdelimumab); TheraCim hR3 (Nimotuzumab); Omnitarg (Pertuzumab, 2C4); Osidem® (IDM-I); OvaRex® (B43.13); Nuvion® (visilizumab); Cantuzumab mertansine (huC242-DMl); NeoRecormon® (Epoetin beta);10487-W001-SECNeumega® (Oprelvekin); Neulasta® (Pegylated filgastrim, pegylated G-CSF, pegylated hu-Met- G-CSF); Neupogen® (Filgrastim); Orthoclone OKT3® (Muromonab-CD3), Procrit® (Epoetin alfa); Remicade® (Infliximab), Reopro® (Abciximab), Actemra® (a nti-l L6 Receptor mAb), Avastin® (Bevacizumab), HuMax-CD4 (zanolimumab), Rituxan® (Rituximab); Tarceva® (Erlotinib); Roferon-A®-(lnterferon alfa-2a); Simulect® (Basiliximab); StelaraTM (Ustekinumab); Prexige® (lumiracoxib); Synagis® (Palivizumab); 146B7-CHO (anti-IL15 antibody, see US7153507), Tysabri® (Natalizumab); Valortim® (MDX-1303, anti-B. anthracis Protective Antigen mAb); ABthraxTM; Vectibix® (Panitumumab); Xolair® (Omalizumab), ETI211 (anti-MRSA mAb), IL-I Trap (the Fc portion of human IgGl and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGFTrap (Ig domains of VEGFRl fused to IgGl Fc), Zenapax® (Daclizumab); Zenapax® (Daclizumab), Zevalin® (Ibritumomab tiuxetan), Zetia (ezetimibe), Atacicept (TACI-lg), anti-a4|37 mAb (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti-CD23 mAb (lumiliximab); BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); SimponiTM (Golimumab); Mapatumumab (human anti-TRAIL Receptor-1 mAb); Ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (Volociximab, anti-a5|31 integrin mAb); MDX-010 (Ipilimumab, anti-CTLA-4 mAb and VEGFR-I (IMC-18F1 ); anti-BR3 mAb; anti-C. difficile Toxin A and Toxin B C mAbs MDX-066 (CDA-I) and MDX-1388); anti-CD22 dsFv-PE38 conjugates (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLP antibodies; anti-TSLP receptor antibody (US8101182); anti-TSLP antibody designated as A5 (US7982016); (anti-CD3 mAb (NI-0401 ); Adecatumumab (MT201 , anti-EpCAM-CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAbs); MDX-1333 (anti- IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti-CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen (FG-3019); anti-CTLA4 mAb; anti-eotaxinl mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAb; anti-sclerostin antibodies (see, US8715663 or US7592429) anti-sclerostin antibody designated as Ab-5 (US8715663 or US7592429); anti- ganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM-CSF Receptor mAb (CAM- 3001); anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNa mAb); anti-IGFIR mAb; anti-IGF-IR mAb (HuMax-Inflam); anti-ILI 2 / IL23p40 mAb (Briakinumab); anti-IL-23p19 mAb (LY2525623); anti-IL13 mAb (CAT-354); anti-IL-17 mAb (AIN457); anti-IL2Ra mAb (HuMax-TAC); anti- 1 L5 Receptor mAb; anti-integrin receptors mAb (MDX-O18, CNTO 95); anti- 1 PIO Ulcerative Colitis mAb (MDX- 1100); anti-LLY antibody; BMS-66513; anti-Mannose Receptor / hCGp mAb (MDX-1307); anti-mesothelin dsFv-PE38 conjugate (CAT-5001 ); anti-PdlmAb (MDX-1 106 (ONO- 4538)); anti-PDGFRa antibody (IMC-3G3); anti-TGFp mAb (GC-1008); anti-TRAIL Receptor-210487-W001-SEC human mAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR / Flt-1 mAb; anti- ZP3 mAb (HuMax-ZP3); NVS Antibody #1 ; NVS Anti body #2; and an amyloid-beta monoclonal antibody.

[0048] Examples of therapeutic proteins suitable for the methods include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, bemarituzumab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261 , dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maridebart cafraglutide, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab,10487-W001-SEC omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarlatamab, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox. Antibodies also include adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezepelumab, and trastuzumab, and antibodies selected from Table G of International Patent Application Publication No. WG2022 / 061092.

[0049] The following examples are given merely to illustrate the present invention and not in anyway to limit its scope.EXAMPLESEXAMPLE 1

[0050] This example describes initial attempts to quantify DTPA in a sample of a pharmaceutical formulation.

[0051] Quantification of DTPA is challenging, because DTPA does not contain a chromophore detectable by direct UV detection. However, there is a need for precise10487-W001-SEC quantification methods, as DTPA is often included in pharmaceutical formulations for its ability to chelate trace heavy metals and inhibit or slow metal-mediated degradation of therapeutic proteins in pharmaceutical formulations.

[0052] Study 1

[0053] An initial attempt to quantify DTPA through a chromatographic method was carried out based on a prior method used to quantify EDTA. In this method, a copper solution is added to a sample comprising EDTA to obtain copper-EDTA metallocomplexes, and the metallocomplexes are chromatographically separated by HPLC. The chromatographic data is collected and the data permits quantification of EDTA. In this study, a copper solution was added to a sample comprising DTPA and then chromatographically separated to obtain chromatographic data. As a positive control, a copper solution was added to a sample comprising EDTA and then chromatographically separated in the same manner as for the DTPA sample. A blank sample comprising water (and lacking DTPA and EDTA) was combined with the copper solution as a negative control. Exemplary results are shown in Figure 1A. As shown in this figure, the chromatographic data forthe sample comprising DTPA was the same as for the blank sample. In both cases, no peaks were observed. In contrast, a distinct peak was observed forthe positive control comprising EDTA. These results suggested that this chromatographic method using the copper solution could not detect DTPA and thus could not be used to quantify DTPA.

[0054] Study 2

[0055] In another initial attempt to quantify DTPA, a chromatographic method was carried out based on a different prior method used to quantify EDTA. In this method, an iron solution is added to a sample comprising EDTA to obtain iron-EDTA metallocomplexes, and the metallocomplexes are chromatographically separated by HPLC. The chromatographic data is collected and the data permits quantification of EDTA. In this study, an iron solution was added to a sample of a pharmaceutical formulation comprising DTPA and then chromatographically separated to obtain chromatographic data. As a positive control, an iron solution was added to a reference standard comprising a known concentration of DTPA (DTPA Standard) and then chromatographically separated in the same manner as for the pharmaceutical formulation sample. Exemplary results are shown in Figure 1 B. As shown in this figure, the chromatogram of the pharmaceutical formulation sample contained high amounts of matrix interference peaks that prevented reliable detection of DTPA in the sample. Thus, this method was deemed not useful for detecting and quantifying DTPA.10487-W001-SEC

[0056] Study 3

[0057] Another initial attempt to quantify DTPA was carried out, and this method was designed based on the conventional EDTA methods and methods described in, e.g., Wang et al., 2016, supra. The method was carried out using a pharmaceutical formulation comprising an lgG4 antibody and DTPA (0.020 mM), in addition to other excipients. Briefly, reference standards each comprising a known concentration of DTPA (DTPA Standards) and pharmaceutical formulation samples were prepared, and then combined with a metallocomplexing agent solution comprising ferric chloride. Each of the DTPA standards and a pharmaceutical formulation sample were applied to a reversed phase liquid chromatography (RPLC) column. During elution, chromatographic data were obtained and such data for the pharmaceutical formulation sample were compared to the chromatographic data of the DTPA standards.

[0058] Preparation of DTPA standards: Five DTPA standards ranging from 0.0025 mM DTPA to 0.0229 mM DTPA were prepared from a diluted DTPA stock solution. A 2.54 mM stock solution of DTPA was first prepared by dissolving 0.100 g of DTPA into 100 mL of Milli-Q water in a 100 mL volumetric flask. An aliquot of this stock solution was then diluted with Milli-Q water to a final concentration of 0.025 mM DTPA. Using the 0.025 mM DTPA solution and water, five DTPA standards were made each having a different concentration within the range of 2.5 uM DTPA to 22.9 uM DTPA. Table 1 A lists the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent (water) added and the known DTPA concentration of the stock solution) of each DTPA standard prepared in this study.

[0059] Preparation of pharmaceutical formulation sample: A sample of a pharmaceutical formulation comprising an lgG4 antibody, 0.020 mM DTPA, and other excipients was prepared by diluting an aliquot of the pharmaceutical formulation two-fold with Milli-Q water. Table 1 A provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of diluent (water) added and the known DTPA concentration of the pharmaceutical formulation prior to dilution) of the pharmaceutical formulation sample prepared in this study (PF Sample 1 ).TABLE 1A10487-W001-SECPF, pharmaceutical formulation

[0060] Addition of Complexing Agent: Each of the DTPA standards and the pharmaceutical formulation sample listed in Table 1 A was mixed 1 :1 with a 0.005% (w / v) ferric chloride solution (pH adjusted to 3.3).

[0061] HPLC Analysis: After being combined with the ferric chloride solution, each of the DTPA standards and the pharmaceutical formulation sample was applied to a Discovery C18 HPLC column (150 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1100 Model HPLC system for HPLC analysis. The HPLC system comprised a quaternary pump, an autosampler, a column thermostat, and a variable wavelength detector (VWD). After the DTPA standards and the pharmaceutical formulation sample were applied to the column, isocratic elution was carried out with 19 mM tetrabutylammonium phosphate (pH 6.5) with 20% (v / v) acetonitrile. Detection was carried out with the VWD at 260 nm. The run time was 10 minutes, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software.Table 1 B details HPLC parameters used in this study.TABLE 1 B

[0062] Exemplary results are shown in Figures 1 C, which is an enlarged view of the boxed region of the spectra in the upper right corner. Peaks for DTPA Standard 1 (having the lowest expected DTPA concentration of 2.5 uM) and DTPA Standard 5 (having the highest expected ETPA concentration of 22.9 uM) were observed and clearly resolved, and peak area correlated with DTPA concentration (Figure 1 C). The peak for the pharmaceutical formulation sample was also observed, and, as shown in Figure 1 C, its peak area was greater than the peak area of DTPA Standard 1 and less than the peak are of DTPA Standard 5, as expected.10487-W001-SECEXAMPLE 2

[0063] This example characterizes the ability of a chromatographic method to quantify DTPA.

[0064] The results of Example 1 suggested that the method of Study 3 could detect DTPA in the pharmaceutical formulation sample. In this study, a method representing a slightly modified method of Study 3 was carried out. In this study, pharmaceutical formulation samples were spiked with a series of DTPA standards (to obtain DTPA-spiked pharmaceutical formulation samples) to determine how well the method quantifies DTPA in a pharmaceutical formulation.

[0065] Preparation of DTPA standards: Five DTPA standards ranging from 0. 5 uM DTPA to 5.0 uM DTPA were prepared from a diluted DTPA stock solution. A 2.54 mM stock solution of DTPA was first prepared as described in Example 1 , and an aliquot of this stock solution was then diluted with Milli-Q water to a final concentration of 0.025 mM DTPA. Using the 0.025 mM DTPA solution and water, five DTPA standards were made, each having a different concentration within the range of 0.5 uM DTPA to 5.0 uM DTPA. Table 2A provides the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent (water) added and the known concentration of DTPA in the stock solution) of each DTPA standard prepared in this study.

[0066] Preparation of pharmaceutical formulation samples: A sample of a pharmaceutical formulation comprising an lgG4 antibody, 0.020 mM DTPA, and other excipients was prepared by diluting an aliquot of the pharmaceutical formulation 50-fold with Milli-Q water, so that the DTPA concentration of this diluted sample was about 0.4 uM. DTPA standards were then spiked into an aliquot of the diluted sample to obtain a series of DTPA-spiked samples ranging in DTPA concentration. As a control, one pharmaceutical formulation sample was not spiked with any DTPA standard (Sample 6). Table 2A provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of diluent (water) added and the known DTPA concentration and volume of the DTPA Standards used to spike) of the spiked pharmaceutical formulation samples and the unspiked control sample (Sample 6) prepared in this study. Samples were prepared in triplicate.TABLE 2A10487-W001-SEC

[0067] Addition of Complexing Agent: Each of the DTPA standards and pharmaceutical formulation samples listed in Table 2A was mixed 1 :1 with a 0.005% (w / v) ferric chloride solution (pH adjusted to 3.3).

[0068] HPLC Analysis: After being combined with the ferric chloride solution, each of the DTPA standards and pharmaceutical formulation samples listed in Table 2A was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1290 Model UHPLC system for HPLC analysis. The UHPLC system comprised a binary pump, an autosampler, a column thermostat, and a variable wavelength detector (VWD). Isocratic elution was carried out with 19 mM tetrabutylammonium phosphate (pH 6.5) with 20% (v / v) acetonitrile. Detection was carried out with the VWD at 260 nm. The run time was 7 minutes, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. Table 2B details HPLC parameters used in this study.TABLE 2B

[0069] Using the chromatographic data for DTPA Standards 1 -5, a linear regression curve was generated correlating the expected DTPA concentration (Table 2A) and the peak area. Using the linear equation of the linear regression curve, a measured DTPA concentration was10487-W001-SEC determined for each of Samples 1 -6. The recovery percent for each of Samples 1 -6 was then determined by subtracting the measured DTPA concentration from the unspiked DTPA concentration and then dividing it by the expected concentration (DTPA concentration in Table 2A) and multiplying by 100%.

[0070] Exemplary results are shown in Figure 1 D and Table 2C. Figure 1 D is a graph of the % recovery plotted as a function of DTPA spike level. An upper threshold recovery percentage and a lower threshold recovery percentage was calculated using the Horwitz equation based on the 95% confidence interval usingthe Student’s t-test (Horwitz, H, Analytical Chemistry, Evaluation of Analytical Methods Used for Regulation of Food and Drugs, 1982, 54, 67-76). The upper threshold recovery percentage and the lower threshold recovery percentage (across all DTPA spike levels) were 111 % and 88%, respectively. As shown in Figure 1 D, the average recovery percent of the DTPA-spiked pharmaceutical formulation samples were greater than the upper threshold recovery percentage across all spike levels. As shown in Table 2C, the average recovery percent for each of the DTPA-spiked pharmaceutical samples (Samples 1 -6) exceeded the upper threshold recovery percentage (111 %).TABLE 2C

[0071] Taken together, the results of this study revealed that the method of this example overestimated the DTPA concentration in the spiked pharmaceutical formulation samples.EXAMPLE 3

[0072] This example demonstrates a troubleshooting study purposed for identifying possible factors that lead to the overestimation of DTPA concentration.

[0073] The results obtained in Examples 1 and 2 suggested that the chromatographic method of Study 3 could detect DTPA in the pharmaceutical formulation sample but could not accurately quantify DTPA in such samples. This study was aimed at identifying factors that lead to DTPA overestimation.10487-W001-SEC

[0074] Preparation ofDTPA standards: Four DTPA standards ranging from 0.5 uM DTPAto 5.0 uM DTPA were prepared from a diluted DTPA stock solution, as previously described in Examples 1 and 2. Table 3A provides the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent (water) added and the known DTPA concentration of the DTPA stock solution) of each DTPA standard used in this study.

[0075] Preparation of Pharmaceutical Formulation Samples: The pharmaceutical formulation used in Examples 1 and 2 comprised an lgG4 antibody, 0.020 mM DTPA, and other excipients, including mannitol, polysorbate 80 (PS80), sodium citrate, and sodium chloride. In this study, pharmaceutical formulation samples were made without DTPA and lgG4 antibody, and without one of the other excipients (mannitol, PS80, sodium citrate, and sodium chloride) to determine whether one or more of the other excipients of the pharmaceutical formulation interfere with accurate quantification of DTPA.

[0076] Pharmaceutical formulation samples of the “A” series were prepared with PS80, sodium citrate, and NaCl, and did not include any DTPA, lgG4 antibody, or mannitol. Pharmaceutical formulation samples of the “B” series were prepared with mannitol, sodium citrate, and NaCl, and did not include any DTPA, lgG4 antibody, or PS80. Pharmaceutical formulation samples of the “C” series were prepared with mannitol, PS80, and NaCl, but did not include any DTPA, lgG4 antibody, and or sodium citrate. Pharmaceutical formulation samples of the “D” series were prepared with mannitol, PS80 and sodium citrate, but did not include any DTPA, lgG4 antibody, or NaCl. A positive control series of pharmaceutical formulation samples were prepared with all of mannitol, PS80, sodium citrate, and NaCl, but did not comprise any lgG4 antibody or DTPA. A negative control series was prepared with just water and a DTPA standard, and this series did not comprise any of the pharmaceutical formulation excipients and lgG4.

[0077] The samples from each series were then spiked with one of the DTPA Standards to arrive at a series of DTPA-spiked samples ranging in DTPA concentration. Table 3A details the expected DTPA concentration (the DTPA concentration calculated based the known DTPA concentration and volume of the DTPA Standards used to spike) and the excipients present in each of the pharmaceutical formulation samples prepared in this study.TABLE 3A10487-W001-SECNone of the samples or controls listed in the table comprised lgG4. The only source of DTPA in each sample or control was DTPA standard.

[0078] Addition of Complexing Agent: Each of the positive controls, negative controls, and pharmaceutical formulation samples listed in Table 3A was mixed 1 :1 with a 0.005% (w / v) ferric chloride solution (pH adjusted to 3.3).

[0079] HPLC Analysis: After being combined with the ferric chloride solution, each of the pharmaceutical formulation samples, positive controls and negative controls listed in Table 3Awas applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1260 Model HPLC system for HPLC analysis. The HPLC system comprised a binary pump, autosampler, column thermostat, refractive index detector (RID), and diode array detector (DAD). Elution was carried out with 19 mM tetrabutylammonium phosphate (pH 6.5) with 20% (v / v) acetonitrile. Detection was carried out with the VWD at 260 nm. The run time was 7 minutes, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. Table 3B details the HPLC parameters used in this study.TABLE 3B10487-W001-SEC

[0080] Exemplary results are shown in Figure 2 which is a graph showing peak area plotted as a function of DTPA concentration for each series. As shown in this figure, the peak areas across all DTPA concentrations overlap for all series, except for Series C (lacking sodium citrate) and the negative control series. These data suggest that the removal of sodium citrate from the pharmaceutical formulation samples of the C series impacted the peak area of the DTPA standards. While the peak areas for the samples comprising lower DTPA concentrations (DTPA Standards 1 and 2) were similar among all series (including Series C), a divergence of peak areas across the series was observed at the higher DTPA concentrations (DTPA Standards 3 and 4). Since a decrease in peak area was not observed with Series D (lacking sodium chloride), it was surmised that the sodium ion did not appearto impact peak area of DTPA. Taken together, these results suggest that the presence of citrate in the pharmaceutical formulation samples impairs accurate quantification of DTPA, leading to overestimation of DTPA in the samples.EXAMPLE 4

[0081] This example demonstrates further analyses of citrate as a possible factor that impacts DTPA quantification.

[0082] The results of Example 3 suggested that the presence of citrate in the pharmaceutical formulation impacts DTPA quantification, as samples without sodium citrate exhibited chromatographic peak areas that were different from the samples containing citrate. It was hypothesized that the presence of citrate caused inaccurate quantitation (e.g., overestimation) of DTPA. To explore this hypothesis, HPLC analysis was carried out with samples comprising different DTPA concentrations made in water, sodium citrate, or a formulation buffer comprising sodium citrate.10487-W001-SEC

[0083] Preparation of 20 mM Sodium Citrate and Formulation Buffer:

[0084] A 20 mM sodium citrate solution was prepared from a sodium citrate stock solution. The sodium citrate stock solution was prepared by dissolving 5.88 g of sodium citrate dihydrate into 800 ml of Milli-Q water. The solution was then adjusted to pH 6.0 with 0.1 M citric acid solution (which was prepared by dissolving 2.10 g of citric acid monohydrate into 100 mL Milli-Q water in a 100 mL volumetric flask). After the pH was adjusted, the sodium citrate stock solution was further diluted to 1 L to obtain a final concentration of 20 mM sodium citrate.

[0085] The pharmaceutical formulations used in Examples 1 and 2 comprised an lgG4 antibody, 0.020 mM DTPA, mannitol, polysorbate 80 (PS80), sodium citrate, and sodium chloride. In this study, a formulation buffer was made without DTPA and the lgG4 antibody and with 20 mM citrate, 3% (w / v) mannitol, 0.02% (w / v) PS80, and 50 mM sodium chloride.

[0086] Preparation of DTPA Samples

[0087] In this study, four series of DTPA samples were prepared using water, phosphate buffer, sodium citrate, or the formulation buffer comprising sodium citrate. The DTPA samples of Series E comprised DTPA at a concentration from 0.0005 mM DTPA to 0.0050 mM and each DTPA sample was prepared with water. Briefly, a 2.54 mM stock solution of DTPA was prepared by dissolving 0.100 g of DTPA into 100 mL of Milli-Q water in a 100 mL volumetric flask. An aliquot of the stock solution was furthered diluted to 0.025 mM DTPA using Milli-Q water. The DTPA samples were prepared using the 0.025 mM DTPA solution.

[0088] The DTPA samples of Series F comprised DTPA at a concentration ranging from 0.0005 mM DTPA to 0.0050 mM DTPA and each DTPA sample was prepared with 20 mM sodium citrate (pH 6), instead of water. Briefly, each DTPA solution was prepared by diluting the 0.025 mM DTPA solution described for Series E with 20 mM sodium citrate to the desired DTPA concentration (0.0005 mM to 0.0050 mM).

[0089] The DTPA samples of Series G comprised DTPA at a concentration from 0.0005 mM DTPA to 0.0050 mM DTPA and each DTPA sample was prepared with a formulation buffer (pH 6) comprising 20 mM sodium citrate, 3% (w / v) mannitol, 0.02% (w / v) PS80, and 50 mM sodium chloride. Each DTPA solution was prepared by diluting the 0.025 mM DTPA solution described for Series E with the formulation buffer to the desired DTPA concentration (0.0005 mM to 0.0050 mM).10487-WG01-SEC

[0090] The DTPA samples of Series H comprised DTPA at a concentration from 0.0005 mM DTPA to 0.0050 mM DTPA and each DTPA sample was prepared with a phosphate buffer (pH 6) comprising 20 mM sodium phosphate, wherein the pH was adjusted to pH 6 with phosphoric acid. Each DTPA solution was prepared by diluting the 0.025 mM DTPA solution described for Series E with the phosphate buffer to the desired DTPA concentration (0.0005 mM to 0.0050 mM).

[0091] Table 4A details each series of samples prepared in this study.TABLE 4A10487-W001-SECExpected DTPA concentration (the DTPA concentration calculated based on the known DTPA concentration and volume of the DTPA Standards used to spike).

[0092] Addition of Complexing Agent: Each of the samples of Series E-H listed in Table 4A was mixed 1 :1 with a 0.005% (w / v) ferric chloride solution (pH adjusted to 3.3).

[0093] HPLC Analysis: After being combined with the ferric chloride solution, each of the DTPA samples was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm;Millipore-Sigma) fitted to an Agilent 1260 Model HPLC system for HPLC analysis. The HPLC system comprised a quaternary pump, autosampler, column thermostat, refractive index detector (RID) and variable wavelength detector (VWD). Elution was carried out with 19 mM tetrabutylammonium phosphate (pH 6.5) with 20% (v / v) acetonitrile. Detection was carried out at 260 nm, the run time was 7 minutes, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. Table 4B summarizes HPLC parameters used in this study.TABLE 4B

[0094] Exemplary results are shown in Figure 3. As shown in this figure, the peak areas for samples of Series F and G (comprising sodium citrate) were aligned with one another, and the peak areas Series E and H (without sodium citrate) were aligned with one another. However, the peak areas for Series F and G did not align with the peak areas for Series E and H. The divergence of the peak areas among the samples was prominent at the two highest DTPA concentrations. The samples of Series E and H (without sodium citrate) at the two highest DTPA concentrations were decreased, relative to the samples of Series F and G (comprising sodium citrate) comprising the two highest DTPA concentrations. Taken together, these results strongly support that the peak area of the samples is impacted by citrate.10487-W001-SECEXAMPLE 5

[0095] This example demonstrates a comparison of two chromatographic methods for quantifying DTPA in a pharmaceutical formulation comprising a protein, DTPA, and citrate.

[0096] The results of Examples 3 and 4 support that quantification of DTPA is impacted when citrate is present. It was hypothesized that both citrate and DTPA were forming complexes with the iron of the ferric chloride solution, and both the citrate-iron complexes and the DTPA- iron complexes were erroneously counted as DTPA-iron complexes to quantify DTPA, thus resulting in an overestimation of the DTPA. To test this hypothesis, two methods for quantifying DTPA were designed: one method which used water as a diluent in DTPA containing solutions and another method which used sodium citrate as a diluent in DTPA containing solutions. Both methods are described below.

[0097] The first method, which was the same as the method of Study 3 in Example 1 , was carried out as follows.

[0098] Preparation of DTPA standards: Five DTPA standards ranging from 0.0025 mM DTPA to 0.0229 mM DTPA were prepared from a diluted DTPA stock solution. A 2.54 mM stock solution of DTPA was first prepared as described in Example 1 , and an aliquot of this stock solution was then diluted with Milli-Q water to a final concentration of 0.025 mM DTPA. Using the 0.025 mM DTPA solution and water, five DTPA standards were made each having a different concentration within the range of 0.0025 mM DTPA to 0.0229 mM DTPA. Table 5A provides the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent (water) and the known DTPA concentration of the stock solution) of each DTPA standard prepared for the first method.

[0099] Preparation of pharmaceutical formulation sample: Six manufactured lots, each comprising a pharmaceutical formulation comprising an lgG4 antibody and 0.020 mM DTPA, as well as other excipients, were used in the first method. An aliquot of each lot was diluted 2- fold with Milli-Q water. Table 5A provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of diluent (water) added and the known DTPA concentration of the pharmaceutical formulation prior to dilution) of the lot samples prepared for the first method.TABLE 5A10487-W001-SEC

[0100] Addition of Complexing Agent: Each of the DTPA standards and pharmaceutical formulation samples listed in Table 5A was mixed 1 :1 with a 0.005% (w / v) ferric chloride solution (pH adjusted to 3.3).

[0101] HPLC Analysis: After being combined with the ferric chloride solution, each of the DTPA standards and the pharmaceutical formulation samples was applied to a Discovery C18 HPLC column (150 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1290 Model UHPLC system for HPLC analysis. The UHPLC system comprised a binary pump, an autosampler, a column thermostat, and a variable wavelength detector (VWD). Elution was carried out with 19 mM tetrabutylammonium phosphate (pH 6.5) with 20% (v / v) acetonitrile. Detection at 260 nm was carried out with the VWD. The run time was 10 minutes, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. Table 5B details HPLC parameters used in this study.TABLE 5B

[0102] The second method, which used sodium citrate instead of waterwhen preparing the DTPA standards and the pharmaceutical formulation sample, was carried out as follows.

[0103] Preparation of DTPA standards: Five DTPA standards ranging from 0.0005 mM DTPA to 0.0050 mM DTPA were prepared from a diluted DTPA stock solution. A 2.54 mM stock10487-W001-SEC solution of DTPA was first prepared as described in Example 1 , and an aliquot of this stock solution was then diluted with 20 mM sodium citrate to a final concentration of 0.020 mM DTPA. Using the 0.020 mM DTPA solution and 20 mM sodium citrate, five DTPA standards were made each having a different concentration within the range of 0.0005 mM DTPA to 0.0050 mM DTPA. Table 5C summarizes the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent and the known DTPA concentration of the stock solution) of each DTPA standard prepared for the second method.

[0104] Preparation of Lot Samples: Six manufactured lots, each comprising a pharmaceutical formulation comprising an lgG4 antibody and 0.020 mM DTPA, as well as other excipients, were used in the second method. An aliquot of each lot was diluted 10-fold with 20 mM sodium citrate and each had an expected DTPA concentration of 0.002 mM. Table 5C provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of diluent added and the known DTPA concentration of the lots prior to dilution) of the lot samples prepared for the second method.TABLE 5C

[0105] Addition of Complexing Agent: Each of the DTPA standards and lot samples listed in Table 5C was mixed 1 :1 with a 0.005%(w / v) ferric chloride solution (pH adjusted to 3.3).

[0106] HPLC Analysis: After being combined with the ferric chloride solution, each of the DTPA standards and the lot samples was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1290 Model UHPLC system for HPLC analysis. The UHPLC system comprised a binary pump, an autosampler, a column thermostat, and a variable wavelength detector (VWD). Elution was carried out with 24 mM tetrabutylammonium hydroxide containing 0.15% phosphoric acid and 25% (v / v) acetonitrile10487-W001-SEC(Mobile Phase A) for 5 minutes, followed by a 5-minute column cleaning using 50% (v / v) acetonitrile, and a 5-minute column equilibration with Mobile Phase A. The total run time was 15 minutes. Detection was carried out with the VWD at 335 nm, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. Table 5D details HPLC parameters used in this study.TABLE 5D

[0107] Exemplary results comparing the first and second methods are shown in Table 5E. The recovery percent for each sample was derived by first calculating the measured concentration for each sample from the linear regression curve generated from the five DTPA standards. The measured concentration was then divided by the expected concentration (0.002 mM after sample dilution) of each sample and multiplied by 100%. As shown in this table, the percent recovery for the second method (which used citrate as the diluent of the standards and samples) was near 100%. In contrast, the percent recovery for the first method (which used water as the diluent of the standards and samples) was much higher than 100%, ranging from 121 % to 130%. The detection wavelength and other chromatographic parameters of the second method may have contributed to an improved baseline and peak sensitivity, decreased interference from artifact peaks, and prolonged column life.TABLE 5E10487-W001-SEC

[0108] Taken together, these results support the use of citrate in the preparation of both the DTPA standards and the samples when quantifying DTPA in a pharmaceutical formulation comprising citrate.EXAMPLE 6

[0109] This example demonstrates a method of quantifying DTPA in a pharmaceutical formulation.

[0110] The results of Example 5 suggested that DTPA in a pharmaceutical formulation can be accurately quantified using sodium citrate as a diluent when preparing DTPA standards and pharmaceutical formulation samples. In this study, the second method of Example 5 was carried out with a single sample of a pharmaceutical formulation alongside two DTPA standards.

[0111] Preparation of DTPA standards: Two DTPA standards ranging from 0.0005 mM DTPA to 0.0050 mM DTPA were prepared from a diluted DTPA stock solution. A 2.54 mM stock solution of DTPA was first prepared as described in Example 1 , and an aliquot of this stock solution was then diluted with 20 mM sodium citrate to a final concentration of 0.020 mM DTPA. Using the 0.020 mM DTPA solution and 20 mM sodium citrate, two DTPA standards were made each having a different concentration within the range of 0.0005 mM DTPA to 0.0050 mM DTPA. Table 6A summarizes the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent and the known DTPA concentration of the stock solution) of each DTPA standard prepared in this study.

[0112] Preparation of Pharmaceutical Formulation Sample: A pharmaceutical formulation comprising an lgG4 antibody and 0.020 mM DTPA, as well as other excipients, were prepared by diluting 10-fold with 20 mM sodium citrate. The sample had an expected DTPA concentration of 0.002 mM as calculated based on the volume of the diluent added and the known concentration of the pharmaceutical formulation prior to dilution (Table 6A).TABLE 6A10487-W001-SEC

[0113] Addition of Complexing Agent: Each of the DTPA standards and pharmaceutical formulation sample listed in Table 6A was mixed 1 :1 with a 0.005% (w / v) ferric chloride solution (pH adjusted to 3.3.

[0114] HPLC Analysis: After being combined with the ferric chloride solution, each of the DTPA standards and the pharmaceutical formulation sample was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1200 Model HPLC system for HPLC analysis. The HPLC system comprised a binary pump, an autosampler, a column thermostat, and a variable wavelength detector (VWD). Elution was carried out with 24 mM tetrabutylammonium hydroxide containing 0.15% phosphoric acid and 25% (v / v) acetonitrile (Mobile Phase A) for 5 minutes, followed by a 5-minute column cleaning using 50% acetonitrile, and a 5-minute column equilibration with Mobile Phase A. The total run time was 15 minutes. Detection was carried out with the VWD at 335 nm, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. Table 6B summarizes HPLC parameters used in this study.TABLE 6B

[0115] Exemplary results are shown in Figure 4. As shown in this figure, the peaks forthe DTPA standards and sample were clearly resolved. The peak area correlated with DTPA concentration. As expected, the peak area of Sample 1 was greater than the peak area for DTPA Standard 1 and less than the peak area for DTPA Standard 5. The results demonstrated that the second method of Example 5 reliably detects and quantifies DTPA in standards and samples, and that the method can be used for DTPA quantification of pharmaceutical formulations comprising citrate.10487-W001-SECEXAMPLE 7

[0116] This example demonstrates an evaluation of the amount of metal in the complexing agent used when quantifying DTPA.

[0117] The results of the prior examples support that DTPA can be reliably quantified from a pharmaceutical formulation comprising citrate, when both the DTPA standards and pharmaceutical formulation samples are prepared using sodium citrate. In this study, the amount of metal in the complexing agent was varied while the amount of sodium citrate used in the preparation of the DTPA standards and pharmaceutical formulation samples remained the same.

[0118] Briefly, three pharmaceutical formulation samples were prepared with 20 mM sodium citrate as described in Example 6. Each of the three samples had an expected DTPA concentration of 0.0050 mM (as calculated based on the volume of diluent added and the known DTPA concentration of the pharmaceutical formulation prior to dilution). Each sample was then mixed 1 :1 with a ferric chloride solution (pH 3.3) comprising (A) 0.005 (w / v)% ferric chloride, (B) 0.010 (w / v)% ferric chloride, or (C) 0.025 (w / v)% ferric chloride. After being combined with the ferric chloride solution, each sample was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1260 Model HPLC system for HPLC analysis. The HPLC system comprised a binary pump, an autosampler, a column thermostat, refractive index detector (RID) and a VWD. Elution was carried out with 24 mM tetrabutylammonium hydroxide containing 0.15% phosphoric acid and 25% (v / v) acetonitrile (Mobile Phase A) for 5 minutes, followed by a 5-minute column cleaning using 50% (v / v) acetonitrile and a 5-minute column equilibration with Mobile Phase A. The total run time was 15 minutes. Detection was carried out with the VWD at 335 nm, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. HPLC parameters used in this study were the same as in Table 6B.

[0119] Exemplary results are provided in Table 7. The max delta peak area was calculated by (A) subtracting the smallest peak area value (0.2303) from the largest peak area value (0.2425) and (B) dividing the difference by the largest peak area and then (C) multiplying by 100%.TABLE 710487-W001-SECCalculated by subtracting the smallest peak area value from the largest peak area value and dividing the difference by the largest peak area value then multiplying by 100%.

[0120] As shown in Table 7, the peak areas were very close to one another in value as the max delta peak area was only 0.0122 (only 5% of the largest peak area). Thus, changing the % ferric chloride solution did not lead to any major shifts in peak area. A peak representing citrate bound to iron is shown in Figure 5, consistent with a peak for citrate bound to iron shown in Figure 4. Taken together, these results support that (1 ) citrate has chelating activity for iron that can be and (2) ferric concentrations as low as 0.005(w / v)% are sufficient to enable reliable measurement of DTPA-ferric complex without disruption from the iron-chelating activity of citrate (when 20 mM sodium citrate is used in preparing the DTPA standards and pharmaceutical formulation samples).EXAMPLE 8

[0121] This example demonstrates measurement of DTPA complexed with iron in the presence of a higher concentration sodium citrate.

[0122] The results of Example 7 support that ferric chloride concentrations of 0.005 (w / v)% and above are sufficient to enable stable measurement of the DTPA-ferric complex, without any disruption from the iron-chelating activity of citrate from 20 mM sodium citrate. In the present study, the study of Example 7 was repeated except that the concentration of sodium citrate used in the preparation of the DTPA standards and pharmaceutical formulation samples was 5x higher than the concentration of the sodium citrate solution used in Example 7.

[0123] Preparation of 100 mM sodium citrate: A 100 mM sodium citrate stock solution was prepared by dissolving 29.4 g of sodium citrate dihydrate into 800 ml of Milli-Q water. The solution was then adjusted to pH 6.0 with the 0.1 M citric acid solution and further diluted to 1 L to obtain a final concentration of 100 mM sodium citrate.

[0124] Preparation of Samples: Three samples each comprising 0.0050 mM DTPA were prepared with 100 mM sodium citrate instead of 20 mM sodium citrate as essentially described in Example 6. Each sample was then mixed 1 :1 with a ferric chloride solution (pH 3.3) comprising (A) 0.005(w / v)% ferric chloride, (B) 0.010(w / v)% ferric chloride, or (C)10487-W001-SEC0.025(w / v)% ferric chloride. After being combined with the ferric chloride solution, each sample was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore- Sigma) fitted to an Agilent 1260 Model HPLC system for HPLC analysis. The HPLC system comprised a binary pump, an autosampler, a column thermostat, refractive index detector (RID) and a VWD. Elution was carried out with 24 mM tetrabutylammonium hydroxide containing 0.15% phosphoric acid and 25% (v / v) acetonitrile (Mobile Phase A) for 5 minutes, followed by a 5-minute column cleaning using 50% (v / v) acetonitrile and a 5-minute column equilibration with Mobile Phase A. The total run time was 15 minutes. Detection was carried out with the VWD at 335 nm, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. HPLC parameters used in this study were the same as in Table 6B.

[0125] Exemplary results are shown in Table 8. As shown in Table 8, the peak area values among the samples were very different, even though the DTPA concentration was the same in each sample. The max delta peak area was 0.0964 (~44% of the largest peak area), which was much higher than that observed in Example 7 (Table 7).TABLE 8Calculated by subtracting the smallest peak area value from the largest peak area value and dividing the difference by the largest peak area value then multiplying by 100%.

[0126] In this example, the citrate concentration of the sodium citrate solution used to prepare the samples was 5x higher (100 mM) than that of the sodium citrate solution used in Example 7 (20 mM). The results obtained in this study suggest that the amount of citrate relative to the amount of iron in the ferric chloride solution matters. When 100 mM sodium citrate was used, the citrate concentration in the samples was too high, given the amount of iron in even the highest % ferric chloride solution. Taken together, these results suggest that the concentration of iron in the ferric chloride solution should be in excess of the citrate concentration in order for the method to reliably quantify DTPA.10487-W001-SECEXAMPLE 9

[0127] This example demonstrates that higher concentration ferric chloride solutions do not negatively impact the quantitation of DTPA when samples and standards are prepared in 20 mM sodium citrate.

[0128] The results of Example 7 support that the peak areas of one DTPA standard complexed with ferric is stable at ferric chloride concentrations of 0.005% and above, and the results of Example 8 support that the amount of citrate relative to the amount of iron in the ferric chloride solution matters when trying to accurately quantify the DTPA in a pharmaceutical formulation comprising DTPA and citrate. The results of Example 8 suggested that iron needs to be in excess of citrate. In this study, we explore the ability to quantify lower DTPA concentrations compared to Examples 7-8 across a range of ferric chloride solutions.

[0129] Preparation of DTPA Standards: A 2.54 mM stock solution of DTPA was prepared by dissolving 0.100 g of DTPA into 100 mL of Milli-Q water in a volumetric flask. An aliquot of the stock solution was furthered diluted to 0.020 mM DTPA using 20 mM sodium citrate. Five DTPA standards (ranging from 0.0005 mM to 0.0050 mM) were prepared using the 0.020 mM solution. Table 9A provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of diluent added and the known DTPA concentration of the stock solution of each DTPA standard prepared in this study.

[0130] Preparation of DTPA-Spiked Pharmaceutical Formulation Samples: Samples of a pharmaceutical formulation comprising an lgG4 antibody, 0.020 mM DTPA, and other excipients were prepared by diluting an aliquot of the pharmaceutical formulation 50-fold with 20 mM sodium citrate, so that the DTPA concentration of this diluted sample was expected as about 0.0004 mM. DTPA standards were then added to an aliquot of the diluted sample to obtain a series of DTPA-spiked samples ranging in DTPA concentration. Table 9A provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of the diluent and the known DTPA concentration and volume of the DTPA Standards used to spike) of the spiked pharmaceutical formulation samples prepared in this study.TABLE 9A10487-W001-SEC

[0131] Addition of Complexing Agent: Each of the DTPA standards and pharmaceutical formulation samples listed in Table 2A was mixed 1 :1 with (A) 0.005 (w / v)% ferric chloride solution, (B) 0.010 (w / v)% ferric chloride or (C) 0.025 (w / v)% ferric chloride.

[0132] HPLC Analysis: After being combined with the ferric chloride solution, each sample was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1200 Model HPLC system for HPLC analysis. The HPLC system comprised a binary pump, an autosampler, a column thermostat, refractive index detector (RID) and a VWD. Elution was carried out with 24 mM tetrabutylammonium hydroxide containing 0.15% phosphoric acid and 25% (v / v) acetonitrile (Mobile Phase A) for 5 minutes, followed by a 5- minute column cleaning using 50% (v / v) acetonitrile and a 5-minute column equilibration with Mobile Phase A. The total run time was 15 minutes. Detection was carried out with the VWD at 335 nm, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. HPLC parameters used in this study were the same as in Table 6B.

[0133] The average spike recovery (%) for each spiked sample and the unspiked control sample was calculated as described in Example 2. Exemplary results are shown in Table 9. As shown in Table 9B, average spike recovery % ranged from 93% to 102% across all samples regardless of the ferric chloride concentration. These results confirm that the revised chromatographic method used to quantify DTPA was reliably quantifies DTPA in the spiked samples, regardless of the ferric chloride concentrations used.TABLE 9B10487-W001-SEC

[0134] This example confirms that the quantitation of DTPA spiked into the mAb sample is not impacted by higher concentrations of ferric chloride.EXAMPLE 10

[0135] This example demonstrates that the quantitation of DTPA when prepared in water does not improve by adding higher concentrations of ferric chloride.

[0136] The results of Example 9 support that the quantitation of DTPA spiked into pharmaceutical formulation-containing samples is not impacted by higher concentrations of ferric chloride when it is prepared in sodium citrate. In this study, it is demonstrated that the quantitation of DTPA spiked into pharmaceutical formulation-containing samples does not improve by adding higher concentrations of ferric chloride.

[0137] Preparation of DTPA Standards: A 2.54 mM stock solution of DTPA was prepared by dissolving 0.100 g of DTPA into 100 mL of Milli-Q water in a volumetric flask. An aliquot of the stock solution was furthered diluted to 0.020 mM DTPA using MiiliQ water. Five DTPA standards (ranging from 0.0005 mM to 0.0050 mM) were also prepared using MiiliQ water. Table 10A provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of diluent added and the known DTPA concentration of the DTPA stock solution) of each DTPA standard prepared in this study.

[0138] Preparation of DTPA-Spiked Pharmaceutical Formulation Samples: A pharmaceutical formulation sample comprising an lgG4 antibody and 0.020 mM DTPA, as well as other excipients, was prepared by diluting an aliquot of the pharmaceutical formulation 50- fold with MiiliQ water, so that the DTPA concentration of this diluted sample was about 0.0004 mM. DTPA standards were then added to an aliquot of the diluted sample to obtain a series of DTPA-spiked samples ranging in DTPA concentration. Table 10A provides the expected DTPA concentration (the DTPA concentration calculated based on the volume of diluent and the known DTPA concentration of the DTPA standards used to spike) of the spiked pharmaceutical formulation samples prepared in this study.10487-W001-SECTABLE 10A

[0139] Addition of Complexing Agent: Each of the DTPA standards and pharmaceutical formulation samples listed in Table 10A was mixed 1 :1 with (A) 0.005 (w / v)% ferric chloride solution, (B) 0.010% ferric chloride or (C) 0.025% ferric chloride.

[0140] HPLC Analysis: After being combined with the ferric chloride solution, each sample was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1200 Model HPLC system for HPLC analysis. The HPLC system comprised a binary pump, an autosampler, a column thermostat, refractive index detector (RID) and a VWD. Elution was carried out with 24 mM tetrabutylammonium hydroxide containing 0.15% phosphoric acid and 25% (v / v) acetonitrile (Mobile Phase A) for 5 minutes, followed by a 5- minute column cleaning using 50% (v / v) acetonitrile and a 5-minute column equilibration with Mobile Phase A. The total run time was 15 minutes. Detection was carried out with the VWD at 335 nm, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. HPLC parameters used in this study were the same as in Table 6B.

[0141] The average spike recovery (%) for each spiked sample and the unspiked control sample was calculated as described in Example 2. Exemplary results are shown in Table 10B. As shown in this table, the overall spike recovery across all DTPA spike levels ranged from 104% to 113%, which is too high. These results strongly support that citrate needs to be used in preparing samples comprising the pharmaceutical formulation to reliably quantify DTPA in a pharmaceutical formulation comprising citrate.10487-W001-SECTABLE 10B

[0142] This example confirms that citrate must be present in the preparation of the DTPA standards as the quantitation of DTPA spiked into the pharmaceutical formulation containing samples did not improve with higher concentrations of ferric chloride.EXAMPLE 11

[0143] This example describes an evaluation of performance parameters for a method of DTPA quantitation.

[0144] Preparation of DTPA standards: DTPA standards ranging from 0.0005 mM DTPA to 0.0036 mM DTPA were prepared from a diluted DTPA stock solution. A 2.54 mM stock solution of DTPA was first prepared as described in Example 1 , and an aliquot of this stock solution was then diluted with 20 mM sodium citrate to a final concentration of 0.020 mM DTPA. Using the 0.020 mM DTPA solution and 20 mM sodium citrate, DTPA standards were made each having a different concentration within the range of 0.0005 mM DTPA to 0.0050 mM DTPA. Table 11 A provides the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent added and the known DTPA concentration of the stock solution) of each DTPA standard prepared in this study.10487-W001-SEC

[0145] Preparation of Pharmaceutical Formulation Sample: Samples comprising a pharmaceutical formulation comprising an lgG4 an lgG4 antibody and 0.020 mM DTPA, as well as other excipients, was prepared by diluting an aliquot of the pharmaceutical formulation 50- fold with 20 mM sodium citrate, so that the DTPA concentration of this diluted sample was about 0.0004 mM. DTPA standards were then spiked into aliquots of the diluted sample to obtain a series of DTPA-spiked samples ranging in DTPA concentration ranging from 0.0005 mM to 0.0036 mMA. Samples were prepared in triplicate (Sample Sets 1 -3). Table 11 A provides the expected DTPA concentrations (the DTPA concentration calculated based on the volume of diluent added and the known DTPA concentration of the DTPA standards used to spike) of each pharmaceutical formulation sample prepared in this study.TABLE 11A

[0146] Addition of Complexing Agent: Each of the DTPA standards and spiked pharmaceutical formulation sample listed in Table 11Awas mixed 1 :1 with a 0.005 (w / v)% ferric chloride solution (pH adjusted to 3.3).

[0147] HPLC Analysis: After being combined with the ferric chloride solution, each of the DTPA standards and the pharmaceutical formulation sample was applied to a Discovery C18 HPLC column (50 mm x 4.6 mm, 5 pm; Millipore-Sigma) fitted to an Agilent 1290 Model UHPLC system for HPLC analysis. The UHPLC system comprised a binary pump, an autosampler, a column thermostat, and a variable wavelength detector (VWD).). Elution was carried out with 24 mM tetrabutylammonium hydroxide containing 0.15% phosphoric acid and 25% (v / v)10487-W001-SEC acetonitrile (Mobile Phase A) for 5 minutes, followed by a 5-minute column cleaning using 50% (v / v) acetonitrile, and a 5-minute column equilibration with Mobile Phase A. The total run time was 15 minutes. Detection was carried out with the VWD at 335 nm, and the chromatographic data were acquired and processed using Chromeleon 7.2.10 software. Table 11 B summarizes HPLC parameters used in this study.TABLE 11 B

[0148] Exemplary results are shown in Table 11 C. The recovery percent for each sample was derived by first calculating the measured concentration for each sample from the linear regression curve generated from the five DTPA standards. The measured concentration was then subtracted from the unspiked concentration and divided by the expected concentration (0.002 mM after sample dilution) of each sample and multiplied by 100%. As shown in Table 11 C, the overall % recovery was 99% and the overall RSD was 2%.TABLE 11 C10487-W001-SECNote: Rounding displayed in the table is for ease of reading. AU calculations were performed in Excel use full precision.

[0149] Linearity

[0150] Linearity was evaluated by determining the correlation of the recovered (measured- unspiked) versus expected concentration of DTPA spiked into a sample comprising a pharmaceutical formulation. The measured versus expected concentrations can be found in Figure 5. The coefficient of determination (R2) was evaluated by plotting the measured versus expected DTPA concentrations from 0.0005 mM to 0.0036 mM. The coefficient of determination (R2) for the trendline was 1 .00 (reference from the R2value 0.9998 in Figure 5, rounded to two decimal places), which met the qualification target of s 0.98.

[0151] Repeatability

[0152] Repeatability was assessed using 6 reported results obtained by preparing 12 replicates of a drug substance (DS) comprising an lgG4 antibody and DTPA at the target concentration of 0.020 mM (before the 10-fold dilution) and injecting each replicate once. The average of each duplicate injection was calculated to yield 6 reported results. The overall %RSD was 1 %, which met the Horwitz equation-based qualification target of s 17% (Table 11 D).Table 11 D: Repeatability of DTPA in DS10487-W001-SEC

[0153] Accuracy

[0154] Accuracy was evaluated using the data from the linearity experiment (above). Method accuracy was calculated by multiplying the slope of the linear equation (1 .0046, referenced from Figure 5) obtained during the five-level spike by 100%. The calculated method accuracy was 100% (rounded to the nearest integer value).

[0155] Range

[0156] The overall method range covered 0.0005 mM to 0.0036 mM with a 10-fold dilution, which was confirmed by acceptable results from the linearity, accuracy, and precision, tests (Table 12 - see below). The qualified range covers an undiluted sample concentration of 0.005 to 0.036 mM.

[0157] Limit of Quantitation (LOQ)

[0158] The sample limit of quantitation (LOQ) was evaluated during the spike recovery experiment by spiking DPTA at 0.0005 mM (or 0.2 ug / mL) into a pharmaceutical formulation sample in triplicate and calculating the average recovery. The average spike recovery of 0.0005 mM (or 0.2 ug / mL) DTPA was 98% as shown in Table 11 C.

[0159] Iron Complexation

[0160] To understand the impact of detecting DTPA upon formation of DTPA-iron metallocomplexes, DTPA standard (0.0050 mM) was combined with orwithout ferric chloride prior to chromatographic separation. DTPA standards were applied to and eluted from an RPLC column as essentially described in Example 5 (second method) or Example 6.Exemplary results are shown in Figure 6. The absorbance signal of DTPA complexed with iron was increased 5.5-fold compared to uncomplexed DTPA.

[0161] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms including the indicated component(s) but not excluding other elements (i.e., meaning “including, but not limited to,”) unless otherwise noted.10487-W001-SEC

[0162] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0163] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0164] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, combinations of the above-described elements in suitable variations thereof are encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

10487-W001-SECWHAT IS CLAIMED:1 . A method of determining a concentration of diethylenetriamene pentaacetate (DTPA) in a pharmaceutical formulation, said method comprising a. providing (i) a reference standard comprising citrate and a known concentration of DTPA and (ii) a sample of a pharmaceutical formulation comprising DTPA, citrate and a protein; wherein the citrate concentration of the reference standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; wherein each of the reference standard and the sample of the pharmaceutical formulation further comprise iron and (i) the iron concentration of the reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and (ii) the iron present in each of the reference standard and sample is in excess of the citrate present in each of the reference standard and sample; b. applying each of the reference standard and the sample of the pharmaceutical formulation to a reversed-phase liquid chromatography (RPLC) column; c. applying a mobile phase to the RPLC column to obtain chromatographic data for the sample and the reference standard; and d. comparing the chromatographic data forthe sample to the chromatographic data forthe reference standard.

2. The method of claim 1 , wherein the citrate present in the reference standard is added and / or the citrate present in the sample of the pharmaceutical formulation is added.

3. The method of any one of the preceding claims, further comprising adding citrate to the reference standard and / or the sample of the pharmaceutical formulation to provide the reference standard and / or the sample of the pharmaceutical formulation of (a).

4. The method of claim 3, comprising adding a citrate solution to the reference standard and / orthe sample of the pharmaceutical formulation.

5. The method of claim 4 wherein the citrate concentration of the citrate solution is the same as the citrate concentration of the pharmaceutical formulation.10487-W001-SEC6. The method of any one of the preceding claims, wherein the citrate concentration of the reference standard and / or the sample of the pharmaceutical formulation is the same as the citrate concentration of the pharmaceutical formulation.

7. The method of any one of preceding claims, wherein the citrate concentration of the citrate solution, reference standard and / or the sample of the pharmaceutical formulation is at least or about 15 mM.

8. The method of claim 7, wherein the citrate concentration of the citrate solution, reference standard and / or the sample of the pharmaceutical formulation is at least or about 20 mM.

9. The method of claim 8, wherein the citrate concentration of the citrate solution, reference standard and / or the sample of the pharmaceutical formulation is at least or about 50 mM.

10. The method of claim 9, wherein the citrate concentration of the citrate solution, reference standard and / or the sample of the pharmaceutical formulation is less than or about 100 mM.11 . The method of any one of the preceding claims, wherein the citrate comprises sodium citrate.

12. The method of any one of the preceding claims, wherein the iron present in the reference standard is added and / or the iron present in the sample of the pharmaceutical formulation is added.

13. The method of any one of the preceding claims, comprising adding iron to the reference standard and / or the sample of the pharmaceutical formulation to provide the reference standard and / or the sample of the pharmaceutical formulation of (a).

14. The method of claim 13, comprising adding an iron solution to the reference standard and / orthe sample of the pharmaceutical formulation.

15. The method of any one of the preceding claims, wherein the iron comprises ferric chloride.

16. The method of any one of claims 13 or 14, wherein the iron concentration of the iron solution is at least or about 0.0005% (w / v) or least or about 0.001 % (w / v).10487-W001-SEC17. The method of claim 16, wherein the iron concentration of the iron solution is at least or about 0.005% (w / v), optionally, wherein the iron solution comprises about 0.005% (w / v) ferric chloride.

18. The method of claim 17, wherein the iron concentration of the iron solution is at least or about 0.010% (w / v), optionally, wherein the iron solution comprises about 0.010% (w / v) ferric chloride.

19. The method of claim 18, wherein the iron concentration of the iron solution is at least or about 0.025% (w / v), optionally, wherein the iron solution comprises about 0.025% (w / v) ferric chloride.

20. The method of any one of the preceding claims, wherein the mobile phase comprises an iron pairing agent.21 . The method of claim 20, wherein the ion pairing agent is tetrabutylammonium hydroxide (TBAH).

22. The method of claim 20 or 21 , wherein the mobile phase comprises about 10 mM to about 50 mM ion pairing agent, optionally, about 20 mM to about 30 mM ion pairing agent.

23. The method of any one of claims 20 to 22, wherein the mobile phase further comprises phosphoric acid.

24. The method of claim 23, wherein the mobile phase comprises about 0.05% (v / v) to about 0.50% (v / v) phosphoric acid, optionally, about 0.15% (v / v) phosphoric acid.

25. The method of any one of the preceding claims, wherein the mobile phase is applied to the column for less than 10 minutes, optionally, about 5 minutes.

26. The method of any one of the preceding claims, further comprising a column cleaning after (d).

27. The method of claim 26, wherein the column cleaning is carried out with an acetonitrile solution.

28. The method of any one of the preceding claims, further comprising applying an acetonitrile solution to the column after (d).

29. The method of claim 27 or 28, wherein the acetonitrile solution is about 5% (v / v) to about 50% (v / v) acetonitrile10487-W001-SEC30. The method of claim 29, wherein the acetonitrile solution is about 20% (v / v) to about 30% (v / v) acetonitrile.31 . The method of claim 30, wherein the acetonitrile solution is 25% (v / v) acetonitrile.

32. The method of any one of claims 27-31 , wherein the acetonitrile solution is applied to the column for less than 10 minutes, optionally, about 5 minutes.

33. The method of any one of the preceding claims, further comprising a column equilibration after (d), optionally after a column cleaning.

34. The method of claim 33, wherein the column equilibration is carried out with the same mobile phase solution applied to the column in (d).

35. The method of claim 34, wherein the column equilibration is carried out in less than 10 minutes, optionally, about 5 minutes.

36. The method of any one of the preceding claims, comprising a column cleaning followed by a column equilibration, wherein (d), the column cleaning and the column equilibration occurs in less than 30 minutes.

37. The method of any one of the preceding claims, wherein the RPLC column comprises a non-polar stationary phase.

38. The method of claim 37, wherein the non-polar stationary phase comprises C18 hydrocarbons.

39. The method of claim 37 or 38, wherein the non-polar stationary phase comprises particles having a particle size of about 5 um.

40. The method of any one of the preceding claims, wherein the RPLC column has a column length of at least or about 50 mm.41 . The method of any one of the preceding claims, wherein the RPLC column has an inner diameter of about 4.6 mm.

42. The method of any one of the preceding claims, wherein the temperature of the RPLC column is greater than 30 degrees C.

43. The method of claim 42, wherein the temperature of the RPLC column is about 40 degrees C.

44. The method of any one of the preceding claims, wherein the flow rate is 0.5 mL / min10487-W001-SEC45. The method of any one of the preceding claims, wherein RPLC column is connected to a system that comprises a detector, optionally, a diode array detector.

46. The method of claim 45, wherein the detector detects at a detection wavelength greater than 260 nm.

47. The method of claim 46, wherein the detector detects at a detection wavelength greater than 300 nm.

48. The method of claim 47, wherein the detector detects at a detection wavelength of 335 nm.

49. The method of any one of the preceding claims, wherein the injection volume is 10 pL.

50. The method of any one of the preceding claims, comprising a. adding a citrate solution to a series of reference standards, wherein each reference standard comprises a known DTPA concentration which is unique among the series of reference standards, whereupon the citrate concentration of each reference standard is the same as the citrate concentration of the sample of the pharmaceutical formulation; b. adding an iron solution to each reference standard of the series, whereupon (i) the iron concentration of each reference standard is the same as the iron concentration of the sample of the pharmaceutical formulation and (ii) the iron present in each reference standard and the sample is in excess of the citrate present in each reference standard and the sample c. applying each reference standard and the sample to the RPLC column; d. applying a mobile phase to the RPLC column to obtain chromatographic data for the sample and the series of reference standards; and e. comparing the chromatographic data forthe sample to the chromatographic data forthe series of reference standards.51 . The method of claim 50, wherein the series comprises a reference standard comprising a minimum DTPA concentration and a reference standard comprising a maximum DTPA concentration, wherein the maximum DTPA concentration is at least 2 times the minimum DTPA concentration.10487-W001-SEC52. The method of claim 51 , wherein the maximum DTPA concentration is at least 5 times the minimum DTPA concentration.

53. The method of claim 52, wherein the maximum DTPA concentration is at least 10 times the minimum DTPA concentration.

54. The method of any one of claims 50 to 53, wherein the minimum DTPA concentration is about 0.0005 mM or about 0.0025 mM.

55. The method of any one of the preceding claims, wherein the chromatographic data comprises peak area and the method comprises comparing the peak area of a peak of the sample to the peak area of a peak of the reference standard.

56. The method of any one of the preceding claims, wherein the chromatographic data comprises peak area and the method comprises creating a standard curve which correlates the peak area of the peak of each reference standard to the known amount of DTPA of the reference standard.

57. The method of any one of the preceding claims, wherein the limit of quantitation (LOQ) of the method is less than 1 pg / mL.

58. The method of claim 57, wherein the LOQ is less than 0.5 pg / mL, optionally, less than 0.25 pg / mL.

59. The method of claim 58, wherein the LOQ is about 0.2 pg / mL.

60. The method of any one of the preceding claims, wherein adding the citrate solution to the sample dilutes the DTPA concentration of the pharmaceutical formulation.61 . The method of any one of the preceding claims, wherein the protein is an antibody.

62. The method of any one of the preceding claims, wherein the citrate concentration of the pharmaceutical formulation is greaterthan about 10 mM and less than about 50 mM, optionally, about 20 mM.

Images