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Validation of Protein A chromatography: orthogonal method with size exclusion chromatography validation for mAb titer analysis

  • Elif Sahinkaya ORCID logo , Aylin Ozkan ORCID logo , Nilay Ozdemir ORCID logo , Merve Elif Aslan ORCID logo , Dilan Bicak ORCID logo , Nilufer Cakir ORCID logo , Hatice Oncel ORCID logo and Ece Kok Yetimoglu ORCID logo EMAIL logo
Published/Copyright: May 23, 2025
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 50 Issue 3

Abstract

Objectives

After reviewing the reference product specifications, found that they include the SEC HPLC result but do not specify Protein A chromatography as a test. Although there is no record of reference product specifications, alternative methods are necessary, particularly for routine analysis of biotechnological products.

Methods

The SEC mobile phase consisted of 0.1 M sodium phosphate and 0.2 M saline buffer pH 6.80 run isocratically at a flow rate of 0.5 mL/min. The Protein A chromatography column was equilibrated (60 mM sodium phosphate buffer, pH 7.4 (binding buffer), 60 mM sodium phosphate buffer, pH 2.5 (elution buffer) gradient system) at 1 mL/min.

Results

The SEC HPLC method validation study was conducted, ranging from (0.01–2.0 mg/mL) 1.0 % (LLOQ level) to 200 %. The Protein A chromatography method between (0.0078 and 4.0 mg/mL) 0.78 % (LLOQ level) and 400 % linearity levels. In the linearity study, both ANOVA and Graphpad results were shown. Both bioanalytical methods were performed by establishing validation parameters for the mAb reference product in accordance with the ICH M10 guideline.

Conclusions

Additionally, the findings shed light on routine titer analysis of biotechnological products, as there is no documentation in the literature regarding Protein A chromatography validation.

Keywords: monoclonal antibody; bioanalytical method development; bioanalytical method validation; biotechnology; chromatography; ICH M10 guideline

Introduction

Monoclonal antibodies (mAbs) are entirely adopted as precious treatment options for hematology, dermatology and oncology patients due to their high therapeutic impact and low side effects [1], 2].

Monoclonal antibodies are typically produced using genetically engineered mammalian cell line expression systems to produce mAbs under controlled growth conditions in bioreactors or flasks [3]. Accurate measurement of mAb concentration or titer is crucial to control product quality during manufacturing and ensure the final product meets specifications/acceptance criterias [4].

The bioanalytical method used in biopharmaceutical quality control (QC) for monoclonal antibody (mAb) aggregate and fragment analysis is size exclusion chromatography. The main advantage of SEC HPLC is the smooth elution conditions that allow protein characterization with minimal effect on conformational structure. Applications of SEC HPLC analysis in the characterization of monoclonal antibodies have been recently reported [5].

Protein A chromatography is a commonly used analytical method to measure mAb titers, based on the specific binding of antibodies to Staphylococcus aureus Protein A [6], 7]. Protein A binds with high affinity and specificity to the crystallizable fragment (Fc) region of immunoglobulins such as IgGs [8]. In the Protein A chromatography titer analytical procedure, mAbs selectively bind to Protein A ligands that are covalently immobilized onto a chromatographic support such as a resin or monolith.

Therefore, it is beneficial to develop and implement comprehensive high-performance liquid chromatography, which maintains the biotechnological product using two distinct separation techniques [9]. Ideally, the retention times at the HPLC separations are completely independent (“orthogonal”). Given that high-performance liquid chromatography stands as the most significant liquid phase separation technique to date, LC × LC stands as a primary contender for separations, as long as the two dimensions of separation remain distinct. Examples of successful LC-LC applications include liquid chromatography in combination with size exclusion chromatography (LC-SEC) [10].

Bioanalytical method validation is staminal to control the safety and validity of biopharmaceutical drugs. Regulatory authorities in the pharmaceutical and biopharmaceutical industry have addressed this topic (e.g., International Conference on Harmonization-ICH, Food and Drug Administration-FDA, and European Medicines Agency, EMA) [11].

Background of the FDA Guideline published in 2018 [12]; highlights the importance of bioanalytical method validation parameters such as lower and upper limit of quantification (LLOQ and ULOQ, respectively) or selectivity, which can affect the reliability of bioanalytical methods. The SEC HPLC method bioanalytical method validation study was designed with the latest published ICH M10 guideline [13].

Materials and methods

Sample and reagents

Bristol-Myers Squibb, USA, LOT: ACG0729 provided the reference product stock sample for the bioanalytical method validation of the mAb.

We obtain the organic solvents, such as methanol and ethanol (J.T. Baker, LOT: 2233905871 and 2234605874), from J.T Baker. Reagents i.e., Sodium Dihydrogen Phosphate Monohydrate (Merck in Darmstadt, Germany. LOT: AM680846210), Di Sodium Hydrogen Phosphate Dihydrate (Merck in Darmstadt, Germany. LOT: K53921580201), Disodium Hydrogen Phosphate (Merck in Darmstadt, Germany. LOT: F2221059232), Sodium Chloride (Merck in Darmstadt, Germany. LOT: K53726017), Tri-Sodium Citrate Dihydrate (Merck in Darmstadt, Germany. LOT: AM1855548), Diethylene Triamine Penta Acetic Acid (Sigma-Aldrich, LOT: 1601033), d-Mannitol (Sigma-Aldrich, LOT: WXBD5209V), Polisorbate 80 (Merck in Darmstadt, Germany. LOT: K56096971) and Ortho-phosphoric Acid (Merck in Darmstadt, Germany. LOT: K54477673225) used during the validation studies. All reagents used in validation studies were HPLC grade.

Experimental

Size exclusion chromatography (SEC) and Protein A chromatography HPLC chromatographic parameters

Size exclusion chromatography (SEC) HPLC chromatographic parameters

Bioanalytical method validation for the mAb was developed and validated by using the HPLC equipment from Waters® with a PDA detector with a specific software, Empower® 3 in which the resulting signals were acquired and the obtained chromatograms were processed.

The ionic strength of the mobile phase, expressed according to the application note and column sheet documentation, should be adjusted to minimize secondary interactions between the packaging material and proteins. To determine the effect of mobile phase concentration on the calibration curve, a set of protein standards was analyzed in 50–250 mM sodium chloride, choosing sodium chloride as it is the most commonly used salt in SEC separations. The analytical method was chosen as sodium buffer and pH 6.8 according to both Waters® application note document and the column sheet document [14], 15].

In column selection, long columns and guard columns were preferred because using long columns will enable to obtain better resolution.

The analytical method development study was carried out with three different flow rates to determine the flow rate after mobile phase selection. All results were compared and, based on the USP <129> monograph and USP application note, it was decided to continue with a flow rate of 0.5 mL/min. In addition, in the light of the information on the column sheet, it is explained that the optimum flow rate is 0.5–1.0 mL/min [16], 17].

The column sheet states that when using a high viscosity buffer, it may be necessary to reduce the maximum flow rate (1.2 mL/min) to not exceed the maximum pressure drop. It has a critical in flow rate selection, with the recommendation that a flow rate equal to 25 % of the maximum flow rate should be used when changing the buffer solution [15].

Injection volume trials were carried out as 5–10–20 µL. Samples prepared with deionized water and formulation buffer at a concentration of 1 mg/mL were given to the SEC HPLC system in 3 different injection volumes. Deionized water, mobile phase and formulation buffer were given to the SEC HPLC system in three different injection volumes and the results were compared. When the column sheet documents and USP <129> monograph were examined, it was concluded that an injection volume of 20 µL was chromatographically appropriate [15], 16].

It was investigated whether there was a difference between the chromatograms if the sample was diluted with deionized water or diluted with formulation buffer. When the results were compared for the two diluents, especially at low concentrations (LLOQ), it was decided that the formulation buffer was ideal. Also it was decided to start with two sample concentrations in the method development study. In the sample chromatograms, the main peaks were compared and it was decided to continue with a sample concentration of 1.0 mg/mL according to the peak heights and system suitability parameters.

The developed final SEC HPLC chromatographic method was developed according to the USP <129> monograph and USP application note so that the flow rate, injection volume, column temperature and autosampler temperature are the same. In the decided method studies, the results were evaluated in terms of symmetry factor, signal/noise ratio, peak area, blank peak area and peak shape [16], 17].

The final size exclusion chromatography (SEC) method was carried out on a TSKgel G3000SWXL, a 5.0 µm, 7.8 × 300 mm and TSKgel SWXL guard column with a 7.0 µm, 6.0 × 40 mm, using photodiode array (PDA) detection at 280 nm. The mobile phase included 0.1 M sodium phosphate and 0.2 M saline buffer 6.80, both of which were run isocratically at the flow rate of 0.5 mL/min. Size exclusion chromatography (SEC) column and autosampler temperatures were set at 25 and 5 °C. Injection volume was performed as 20 μL. The chromatographic separation was obtained with retention time of 16.6 min. For all bioanalytical method validation parameters except for specificity/selectivity (70 min) and robustness (flow rate change) parameters, run time was determined 35 min.

Protein A chromatography is a common analytical method for determining monoclonal antibody (mAb) titers due to its high efficiency. Accurate and reliable results of this procedure are imperative, as quantifying the total present for monoclonal antibody (mAb) samples.

In Protein A chromatography initial mobile phase concentration of the method; it was used as 20 mM binding buffer & 20 mM elution buffer according to protein A column sheet document [18].

However, in analytical method development, trials were made according to the column sheet document. While the Protein A chromatography analytical method was being developed to determine the LLOQ level, it was decided to try higher salt concentrations and analyzes were carried out because the symmetry factor of the peak was greater than 2.0.

According to monograph E.P. 2.2.46; the ideal situation is for the symmetry factor to be between 0.8 and 1.5 [19]. However, considering the column sheet and the documents shared by the manufacturers, a maximum symmetry factor of 2.0 is stated as the acceptance criteria. As a result, method development trials were made by gradually increasing the salt concentration in the mobile phase. When the chromatograms were examined one by one at the LLOQ level, the symmetry factor was determined to be lower than 2.0.

The analytical method was studied in terms of carry-over effect due to the peak detected in mobile phase A and formulation buffer. Since the carry-over effect can cause analytical problems, especially during the method validation phase, attempts have been made to increase the injector washing time as an analytical solution.

In the initial analytical method used before the method development studies started, the injector washing time was 6 s. Thereupon, it was thought that the injector washing time of 6 s was not sufficient, and the injector washing time was tried as 12 and 30 s. Formulation buffer (blank) peak areas were compared from the results of the studies. According to the ICH M10 guide; the area of the peak detected upon blank injection should not be greater than 20 % of the area of the LLOQ level [13].

For the method development study, formulation buffer and mobile phase A were tested as sample diluent. It was investigated whether there was a difference between the chromatograms if the sample was diluted with mobile phase A or diluted with formulation buffer. When the results were compared for the two diluents, especially at low concentrations (LLOQ), it was decided that the formulation buffer was ideal.

In addition, carry-over effect results should be studied in validation studies for the highest concentration level of the calibration standard and the study concentration. LLOQ level areas and peak areas obtained from mobile phase A and formulation buffer chromatograms were compared. 30 s injector washing time was selected according to carry-over results.

The final Protein A chromatography method was carried out on a TSKgel Protein A – 5 PW; 4.6 × 35 mm using photodiode array (PDA) detection at 280 nm. The column was equilibrated (60 mM sodium phosphate buffer (binding buffer), pH 7.4 (mobile phase A), 60 mM sodium phosphate buffer, (elution buffer) pH 2.5 (mobile phase B), Gradient system at 1 mL/min and 20 μL of filtered (0.2 μm, Millipore) sample was injected. The chromatographic separation was obtained with retention time of 3.1 min. For all bioanalytical method validation parameters except for specificity/selectivity (20 min) and robustness (flow rate change) parameters, run time was determined 10 min.

Preparation of SEC buffer solution/mobile phase: 0.1 M sodium phosphate and 0.2 M saline buffer were prepared. The pH of this solution was adjusted to 6.80±0.05. Filtered with 0.22 μm filter and degassed.

Preparation of Protein A buffer solution/mobile phase: 60 mM sodium phosphate buffer, pH 7.4 (mobile phase A, binding buffer), 60 mM sodium phosphate buffer, pH 2.5 (mobile phase B, elution buffer) were prepared. The pH of these solutions was adjusted to 7.40±0.05 and 2.50±0.05. Filtered with 0.22 μm filter and degassed.

Preparation of blank/diluent (formulation buffer)

60 g d-mannitol, 16 mg diethylene triamine penta acetic acid, 11.76 g sodium citrate and 5.84 g sodium chloride are precisely weighed and mixed into a 2,000 mL flask containing 1,950 mL deionized water. Add 374 µL polysorbate 80 to the solution and mix thoroughly, completed to volume with deionize water. The pH of this solution is adjusted to 6.0±0.05 with sodium hydroxide or hydrochloric acid. It is filtered with a 0.22 µm filter and degassed.

Preparation of reference product stock sample solution: The reference product was used as a stock solution. Reference product’s concentration is 10 mg/mL (concentration=10.0 mg/mL).

Preparation of sample solution (study sample for Protein A chromatography and SEC HPLC methods): Reference product stock sample solution was diluted with formulation buffer to 1.0 mg/mL (concentration=1.0 mg/mL).

Bioanalytical method validation study

Once the chromatographic and experimental conditions were optimized, the bioanalytical methods were validated by the determination of the following parameters: specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), robustness, and system suitability test, following the International Conference on Harmonisation (ICH) guidelines. ICH M10 guideline was followed when carrying out the bioanalytical validation of SEC analysis for mAb [13].

In order to fulfill bioanalytical method validation requirements, specificity, linearity, range, LLOQ, precision and accuracy, robustness, and solution stability parameters were performed. The SEC HPLC Bioanalytical Method Validation Study applied a specificity test to validate the analytical method’s ability to measure the desired substances (mAb) in a given study sample. The preparation of SEC HPLC solutions for the linearity parameter in the bioanalytical method validation involves serial dilution of the linearity stock solution (200, 100, 50, 25, 5, 2.5, 2.0, 1.0 %). Protein A chromatography solutions were made by slowly diluting a linearity stock solution in order to meet the requirements of the bioanalytical method validation. The solutions were prepared at various concentrations: 400 , 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 %.

Specificity

The main peak in the SEC HPLC chromatogram obtained from the study sample solution met the acceptance criteria of purity angle<purity threshold.

The main in the Protein A chromatography chromatograms obtained from the study sample solution met the acceptance criteria of purity angle<purity threshold.

Carry-over

Formulation buffer, mobile phase A and mobile phase B was injected into the systems after each sample solution injection. The peak area of main peak observed in the formulation buffer solution and mobile phases were detected. Carry-over from formulation buffer solution injections should not be more than 20 % of the LLOQ area of the main peak.

Linearity

The linearity graph was plotted by using the average peak area and the concentrations of solutions having different concentrations. In addition, correlation coefficient, slope, and intercept values were reported. Correlation coefficient was found more than 0.995. Relative standard deviation (RSD) between results were recorded by calculating SD (standard deviation).

Lower limit of quantitation (LLOQ)

LLOQ concentration was determined according to signal to noise (S/N) ratio. Signal to noise ratio of main peak obtained from LLOQ solution injections were found at least 10.0. The relative standard deviation (RSD%) of areas obtained from main peak in the chromatograms obtained from injection performed after LLOQ% level determination was found less than 20.0 %. By checking the S/N values of the chromatograms, the optimal LLOQ level was determined for SEC and Protein A chromatography analyses. The ULOQ level was determined as specified in the ICH M10 guideline [13].

Precision & accuracy

Study samples were prepared by serial dilution method at 200 , 100, 50, 25, 5, 2.5, 2.0, 1.0, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 % levels. Separate vials were prepared for each level prepared and six consecutive injections were performed in the HPLC systems. The standard curve was created. When the standard curve result was found applicable, accuracy and precision levels were prepared. Immediately after for SEC analysis; LLOQ (1.0 % – 0.01 mg/mL), approximately three times the LLOQ level (Low QC) (2.5 % – 0.025 mg/mL), 50.0 % (MID QC) (0.5 mg/mL) and 75.0 % (ULOQ – 0.75 mg/mL).

Immediately after for Protein A chromatography analysis; LLOQ (0.78 % – 0.0078 mg/mL), approximately three times the LLOQ level (Low QC) (3.125 % – 0.03125 mg/mL), 50.0 % (MID QC) (0.5 mg/mL) and 100.0 % (ULOQ – 1.0 mg/mL). Sample solutions were performed separately in five Eppendorf tubes from concentration levels. Precision and accuracy parameters were performed by analyzing four concentration levels in at least three independent analytical runs.

For standard curve, R2 results were found more than 0.995. RSD% results at the LLOQ level were found not greater than 20.0 %. RSD% result at each concentration level were found not greater than 15.0 %. Accuracy analysis results were between 80.0 and 120.0 % for the LLOQ level, between 85.0 and 115.0 % for other levels [13]. All system suitability parameters were found applicable.

Intermediate precision & accuracy

The intermediate precision and accuracy parameter in bioanalytical method validation study was performed to carry out the precision and accuracy study by a different scientist, using a different HPLC equipments and different columns on different days.

Study samples were prepared as in the precision and accuracy parameter. All acceptance criteria and system suitability parameters were found applicable.

Robustness

The robustness of a bioanalytical method is a measure of its ability to remain unaffected by small but stable changes in method parameters and is an indicator of reliability during normal procedure execution by changes such as wavelength, flow rate, mobile phase composition and column temperature. Sample results as well as symmetry factor, and theoretical plate count of the main peak which are the one of the main system suitability parameters weren’t affected adversely by the changes done on the method parameters.

Robustness – solution stability

Solutions whose stabilities are studied should be maintained at 5 °C temperature to evaluate the duration in which they are stable. The sample solution was prepared in accordance with the SEC and Protein A chromatography bioanalytical method, kept at 5 °C for a period of initial, 6–12–24–36–48–72 h analyzed, and the change % was calculated based on the mean area results.

Results and discussions

Specificity

The main peak of the mAb in the sample chromatograms successfully met the purity angle and purity threshold criteria in SEC HPLC and Protein A chromatography analysis.

Carry-over

When mobile phase, formulation buffer and reference product were given to the SEC HPLC system, no peak was detected where the main peak’s retention time. For Protein A chromatography, when formulation buffer and mobile phase A (Binding buffer) were injected into the HPLC system, a peak was detected at the main peak’s retention time, where the main peak is. Therefore, in Protein A chromatography, the HPLC system was controlled by injection of formulation buffer at the highest calibration curve concentration (400 %) and after the study concentration (100 %). When the six injection reference products were injected into the HPLC system, the areas were recorded to check the carry-over effect. The area average of the LLOQ level checked for three days was determined as 13,351. When checking the areas obtained from formulation buffer solution injections, the LLOQ was not found to be more than 20.0 % of the area average. The carry over effect was also found within the limit as indicated in Table 1.

Table 1:

Protein A chromatography carry-over test results table.

Peak name Area Peak name Area
Formulation buffer 1,363 Formulation buffer 1,166
1.0 mg/mL sample solution-1 2,052,427 4.0 mg/mL sample solution-1 7,627,365
Formulation buffer 1,888 Formulation buffer 2,167
1.0 mg/mL sample solution-2 2,056,493 4.0 mg/mL sample solution-2 7,626,967
Formulation buffer 1,567 Formulation buffer 2,401
1.0 mg/mL sample solution-3 2,054,030 4.0 mg/mL sample solution-3 7,623,487
Formulation buffer 1,683 Formulation buffer 2,299
1.0 mg/mL sample solution-4 2,054,519 4.0 mg/mL sample solution-4 7,635,175
Formulation buffer 2,211 Formulation buffer 1,955
1.0 mg/mL sample solution-5 2,049,919 4.0 mg/mL sample solution-5 7,618,362
Formulation buffer 1,918 Formulation buffer 2,089
1.0 mg/mL sample solution-6 2,061,478 4.0 mg/mL sample solution-6 7,632,004
Formulation buffer 1,766 Formulation buffer 2,188

Linearity

We determined the slope, y-intercept, and correlation coefficient determination (R) values. The tables below provide the average area values of the injected and performed linearity levels across three different day trials. Figure 1 provides the linearity graphs for the main peaks.

Figure 1: The linearity graphs for day 1–2–3 average main peak of applied chromatographic methods.
Figure 1:

The linearity graphs for day 1–2–3 average main peak of applied chromatographic methods.

The tables below provide a comparison between Protein A chromatography and SEC HPLC. All results of LLOQ level’ signal/noise values for the days are given in Supplementary Table 1. While Tables 2 and 3 demonstrate linearity day 1–2–3 summary for SEC-HPLC and Protein A respectively, Tables 4 and 5 indicate overall ANOVA analysis of two chromatographic methods. Study concentrations’ chromatographic profiles of two methods obtained for mAb titer analysis in Figure 2. All results show that both bioanalytical methods are linear.

Table 2:

SEC HPLC linearity day-1–2–3 summary results table.

Concentration level (%) Concentration, mg/mL (x) Area (y) Calculated area (ycal)a Change (y − ycal) Square of changes (y − ycal)2
200 2.0 8,158,278 8,171,274 12,995 168,881,100.28
100 1.0 4,110,219 4,082,753 −27,466 754,361,801.77
50 0.5 2,040,985 2,038,492 −2,493 6,214,030.28
25 0.25 1,006,824 1,016,362 9,539 90,987,058.14
5 0.05 193,846 198,658 4,812 23,152,553.16
2.5 0.025 92,783 96,445 3,662 13,412,846.94
2.0 0.020 74,516 76,002 1,487 2,210,594.10
1.0 0.010 37,654 35,117 −2,537 6,434,179.77
RSS 1,065,654,164

  1. aCalculated area: value calculated with the line formula (y=mx±n) by inserting concentration values into “x”.

Table 3:

Protein A chromatography linearity day-1–2–3 summary results table.

Concentration level (%) Concentration, mg/mL (x) Area (y) Calculated area (ycal) Change (y − ycal) Square of changes (y − ycal)2
400 4.0 7,667,988 7,718,351 50,362 2,536,368,352.97
200 2.0 3,952,167 3,862,161 −90,006 8,100,995,157.62
100 1.0 1,962,539 1,934,066 −28,473 810,713,635.80
50 0.5 968,106 970,019 1,913 3,660,069.00
25 0.25 475,699 487,995 12,296 151,192,749.66
12.5 0.125 233,487 246,983 13,496 182,148,866.82
6.25 0.0625 114,246 126,477 12,231 149,599,994.61
3.125 0.03125 55,189 66,224 11,035 121,775,666.73
1.56 0.0156 26,565 36,050 9,485 89,956,079.87
0.78 0.0078 13,351 21,011 7,660 58,674,591.24
RSS 12,205,085,164
Table 4:

mAb SEC HPLC linearity day-1–2–3 summary ANOVA results.

Summary output
Regression statistics
Multiple R 0.99999
R square 0.99998
Adjusted R square 0.99997
Standard error 13,327.00
Observation 8
ANOVA
df SS MS F Significance F
Regression 1 5.78122 × 1013 5.78122 × 1013 325,502.84 1.9571 × 10−15
Residual 6 1,065,654,164 177,609,027.4
Total 7 5.78133 × 1013
Coefficient Standard error t Stat p-Value Lower 95 % Upper 95 %
Intercept −5,767.99 5,841.72 −0.99 0.36 −20,062.2 8,526
X variable 1 4,088,520.85 7,166.20 570.53 1.95712 × 10−15 4,070,985.8 4,106,055
Table 5:

mAb Protein A chromatography linearity day-1–2–3 summary ANOVA results.

Summary output
Regression statistics
Multiple R 0.99989
R square 0.99980
Adjusted R square 0.9998
Standard error 39,059.4
Observation 10
ANOVA
df SS MS F Significance F
Regression 1 5.5562 × 1013 5,5562 × 1013 36,418.91572 6.3616 × 10−16
Residual 8 12,205,085,164 1,525,635,646
Total 9 5.55742 × 1013
Coefficient Standard error t Stat p-Value Lower 95 % Upper 95 %
Intercept 5,971.50 14,756.86 0.40 0.696 −28,057.9 40,000.8
X variable 1 1,928,094.80 10,103.34 190.84 6.3616 × 10−16 1,904,796.4 1,951,393
Figure 2: Chromatographic profile of two methods obtained for mAb titer analysis.
Figure 2:

Chromatographic profile of two methods obtained for mAb titer analysis.

Data analysis summary of linearity parameter

See Tables 2, 3, 4 and 5.

Data analysis summary of precision & accuracy parameter

Protein A chromatography and SEC HPLC comparison results of LLOQ, low QC, MID QC and ULOQ results were given in the Supplementary Tables 2, 3, 4, and 5. All comparison results was graphed with graphpad application. And Figure 3 was given below. All results were found applicable to both bioanalytical methods.

Figure 3: Average recovery results of precision and accuracy parameters for SEC HPLC and Protein A chromatography.
Figure 3:

Average recovery results of precision and accuracy parameters for SEC HPLC and Protein A chromatography.

Data analysis summary of robustness & solution stability parameters

Wavelength change and column temperature change were carried out under the same conditions in Protein A chromatography and the SEC HPLC method. However, since the flow rates of the two methods were different, analyses were carried out as 0.3–0.5–0.7 mL/min in the SEC HPLC method and 0.8–1.0–1.2 mL/min in the Protein A chromatography analysis.

For solution stability change % for the sample solution was found less than 2.0 %. Protein A chromatography and SEC HPLC robustness and solution stability results were given in the Supplementary Tables 6, 7 and 8. All results were found applicable to both bioanalytical methods. Both bioanalytical method’s solution stability as found 72 h.

Conclusions

In this study, the goal was to develop Protein A chromatography as an orthogonal method for mAb titer analysis. We performed validation parameters under the guidance of ICH M10, reporting findings that were accurate, specific, linear, and repeatable.

The range of the bioanalytical method for mAb was determined to be between 0.01 and 2.0 mg/mL (1.0–200 %) for SEC HPLC and between 0.0078 and 4.0 mg/mL (0.78–400 %) for Protein A chromatography. The guideline suggests using validated bioanalytical methods for mAb SEC HPLC and Protein A chromatography analysis.

Both chromatographic methods’ linearity results met the acceptance criteria. The obtained results were analyzed by using ANOVA for the three-day output and found that the adjusted R-square was greater than 0.995 with no significant variation. The regression analysis’s confidence level revealed a negative (−) value for the lower 95 % and a positive (+) value for the upper 95 %. These findings indicated that the graph is linear, and the results were applicable.

According to the ICH M10 guideline, the accuracy and precision analysis results ranged from 80.0 to 120.0 % for the LLOQ level and from 85.0 to 115.0 % for other levels. We found the main peak’s symmetry factor to be lower than 2.0. The main peak’s EP Plate Count value exceeded 2,000.

When all the results were compared, it was concluded that the results were within the specified limits and the system suitability results were applicable.

The intermediate precision and accuracy samples for each study level fell within the specified limits. Similar to the precision and accuracy parameter, we validated all results and found them acceptable.

The robustness results were reported and verified against the acceptance criteria. The results indicated that the small changes in the laboratory have no significant effect. Solution stability results demonstrated that the study sample solution remained stable at 5 °C for 72 h using both bioanalytical methods.

Corresponding author: Prof. Dr. Ece Kok Yetimoglu, Department of Analytical Chemistry, Marmara University, Istanbul, Turkiye, E-mail:

  1. Research ethics: Not applicable.

  2. Informed consent: Not applicable.

  3. Author contributions: The authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: None declared.

  7. Data availability: The raw data can be obtained on request from the corresponding authors.

References

1. Ecker, DM, Jones, SD, Levine, HL. The therapeutic monoclonal antibody market. MAbs 2015;7:9–14.10.4161/19420862.2015.989042Search in Google Scholar PubMed PubMed Central

2. Singh, S, Kumar, NK, Dwiwedi, P, Charan, J, Kaur, R, Sidhu, P, et al.. Monoclonal antibodies: a review. Curr Clin Pharmacol 2003. https://doi.org/10.2174/1574884712666170809124728.Search in Google Scholar PubMed

3. Javanmard, S. An overview of the various appropriate types of cell lines for the production of monoclonal antibodies. Int J Pharm Investig 2023;13:381–92. https://doi.org/10.5530/ijpi.13.3.049.Search in Google Scholar

4. Paradina Fernàndez, L, Calvo, L, Viña, L. Development and validation of an affinity chromatography-protein G method for IgG quantification. Int Sch Res Not 2014;2014:1–7. https://doi.org/10.1155/2014/487101.Search in Google Scholar PubMed PubMed Central

5. Zhang, C, Rodriguez, E, Bi, C, Zheng, X, Suresh, D, Suh, K, et al.. High performance affinity chromatography and related separation methods for the analysis of biological and pharmaceutical agents. Analyst 2018;143:374–91. https://doi.org/10.1039/C7AN01469D.Search in Google Scholar PubMed PubMed Central

6. Tscheliessnig, A, Jungbauer, A. High-performance monolith affinity chromatography for fast quantitation of immunoglobulin G. J Chromatogr A 2009;1216:2676–82. https://doi.org/10.1016/j.chroma.2008.07.063.Search in Google Scholar PubMed

7. Leblebici, P, Leblebici, ME, Ferreira-da-Silva, F, Rodrigues, AE, Pais, LS. Separation of human immunoglobulin G subclasses on a protein A monolith column. J Chromatogr B 2014;962:89–93. https://doi.org/10.1016/j.jchromb.2014.05.029.Search in Google Scholar PubMed

8. Fekete, S, Beck, A, Veuthy, JL, Guillarme, D. J Pharm Biomed Anal 2014.Search in Google Scholar

9. Schoenmakers, PJ, Marriott, P, Beens, J. LC–GC Eur 2003.Search in Google Scholar

10. van der Horst, A, Schoenmakers, PJ. Comprehensive two-dimensional liquid chromatography of polymers. J Chromatogr A 2003;1000. https://doi.org/10.1016/s0021-9673(03)00495-3.Search in Google Scholar PubMed

11. Boulanger, B, Rozet, E, Moonen, F, Rudaz, S, Hubert, P. A risk-based analysis of the AAPS conference report on quantitative bioanalytical methods validation and implementation. J Chromatogr B 2009;877. https://doi.org/10.1016/j.jchromb.2009.06.019.Search in Google Scholar PubMed

12. U.S. Food and Drug Administration. Bioanalytical method validation guidance for industry; 2018.Search in Google Scholar

13. Bioanalytical Method Validation and Study Sample Analysis M10; 24 May 2022.Search in Google Scholar

14. https://www.waters.com/nextgen/us/en/library/application-notes/2011/methoddevelopment-for-size-exclusion-chromatographyofmonoclonalantibodies.Search in Google Scholar

15. https://www.separations.us.tosohbioscience.com/HPLC_Columns/id8327/TSKgel_G3000SWxl.Search in Google Scholar

16. https://www.usp.org/sites/default/files/usp/document/ourwork/biologics/guide-proc-for-recombinant-therapeutic.pdf.Search in Google Scholar

17. https://www.usp.org/sites/default/files/mAb/USP_AggregationAnalysis_ApplicationNote_FINAL.pdf.Search in Google Scholar

18. tskgel®protein a-5pw affinity HPLC column beginners guide for mAb titer analysis-column sheet document.Search in Google Scholar

19. EP 2.2.46 monograph.Search in Google Scholar

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/tjb-2024-0186).

Received: 2024-08-07
Accepted: 2025-02-11
Published Online: 2025-05-23

© 2025 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/tjb-2024-0186
Keywords for this article
monoclonal antibody; bioanalytical method development; bioanalytical method validation; biotechnology; chromatography; ICH M10 guideline
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Review Article
  3. Unveiling the hidden clinical and economic impact of preanalytical errors
  4. Research Articles
  5. To explore the role of hsa_circ_0053004/hsa-miR-646/CBX2 in diabetic retinopathy based on bioinformatics analysis and experimental verification
  6. Study on the LINC00578/miR-495-3p/RNF8 axis regulating breast cancer progression
  7. Comparison of two different anti-mullerian hormone measurement methods and evaluation of anti-mullerian hormone in polycystic ovary syndrome
  8. The evaluation of the relationship between anti angiotensin type I antibodies in hypertensive patients undergoing kidney transplantation
  9. Evaluation of neopterin, oxidative stress, and immune system in silicosis
  10. Assessment of lipocalin-1, resistin, cathepsin-D, neurokinin A, agmatine, NGF, and BDNF serum levels in children with Autism Spectrum Disorder
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  14. Myotonic dystrophy type 1 and oxidative imbalance: evaluation of ischemia-modified albumin and oxidant stress
  15. Antioxidant and alpha-glucosidase inhibitory activities of flavonoids isolated from fermented leaves of Camellia chrysantha (Hu) Tuyama
  16. Examination of the apelin signaling pathway in acetaminophen-induced hepatotoxicity in rats
  17. Integrating network pharmacology, in silico molecular docking and experimental validation to explain the anticancer, apoptotic, and anti-metastatic effects of cosmosiin natural product against human lung carcinoma
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  22. Letter to the Editor
  23. Letter to the Editor: “Gene mining, recombinant expression and enzymatic characterization of N-acetylglucosamine deacetylase”
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