Development of a New Tandem Ion Exchange and ... - ACS Publications

Apr 29, 2019 - Chromatography Method To Monitor Vaccine Particle Titer in Cell. Culture Media. Andrew W. ... being practical methods for direct cell c...
0 downloads 0 Views 990KB Size
Technical Note Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/ac

Development of a New Tandem Ion Exchange and Size Exclusion Chromatography Method To Monitor Vaccine Particle Titer in Cell Culture Media

Anal. Chem. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 04/29/19. For personal use only.

Andrew W. Shaddeau, Nicole A. Schneck,* Yile Li, Vera B. Ivleva, Frank J. Arnold, Jonathan W. Cooper, and Q. Paula Lei* Vaccine Production Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Gaithersburg Maryland United States S Supporting Information *

ABSTRACT: A new tandem chromatography method was developed to directly measure the titers of various vaccine candidate molecules in cell culture without a prior purification step. The method utilized a strong anion exchange chromatography (IEC) column in tandem with a size exclusion chromatography (SEC) column to efficiently separate the nanoparticle and virus-like particle (VLP) vaccine molecules from host cell proteins and other components in the cell culture media. The dual (charge and hydrodynamic size) separation mode was deemed necessary to achieve good separation of the vaccine product for quantitation. The method development and quality assessment illustrated herein was focused on the influenza vaccine candidate H1ssF, a hemagglutinin (group 1) stabilized stem molecule fused to ferritin to form nanoparticles. This newly established method was then successfully applied to several vaccine candidate developmental projects, such as the hemagglutinin−ferritin (HAF) nanoparticle and encephalitic alphavirus VLP-based vaccines. This IEC−SEC strategy was established as a platform approach for direct titer measurement of novel vaccine molecules in cell culture.

V

H1ssF).4,5 Additionally, potential vaccines were developed to protect public health from Western (WEE), Eastern (EEE), and Venezuelan (VEE) equine encephalitic alphaviruses.3 To that end, virus-like particles (VLPs) mimicking the different types of viruses (WEE, EEE, and VEE) but lacking viral nucleic acid were expressed in a mammalian cell line as vaccine candidates (monovalent or trivalent).9,10 In these vaccine development campaigns (H1ssF, seasonal HAF, and encephalitic alphavirus VLPs), scale-up cell culture to produce high titers was essential. A fast and reliable analytical method that can directly measure the vaccine particle titer in cell culture without a prior purification step is valuable for timely support to upstream process development.4,8,11 Unlike antibody development where protein A affinity chromatography typically serves as a universal approach for titer growth determination in cell culture media, efficient analytical chromatography titer measurement has yet to be established for vaccine development.12 Because of the high costs and long lead times, custom antibody columns for each individual vaccine product

accination is an effective strategy to prevent outbreaks of many infectious diseases, including influenza viruses and alphaviruses.1−3 Because of the rapid influenza antigenic drift of hemagglutinin (HA), making efficacious seasonal vaccines for influenza can be challenging, with short development timelines and the potential of missing the target if unexpected pandemic virus strains emerge. Most influenza vaccines target the head region of the full-length HA; however, the highly conserved HA stem region may also potentially be a target for broad and protective immunity.4 Therefore, development of a vaccine that can elicit broader and sustainable immunity compared with that elicited by the traditional influenza vaccine is desired.5,6 In addition to the vaccine design aspect, recombinant protein technology has improved, with higher production capacities and faster development timelines compared with those of the traditional egg-based technology.7,8 Along these lines, two recent influenza vaccine candidates that may induce broader and long-lasting protective immunity were developed by the Vaccine Research Center, which utilized mammalian cell lines for product expression of self-assembled nanoparticles comprising ferritin (F) genetically fused with either (1) full-length seasonal HA (HAF constructs HAF-New Caledonia, HAF-Singapore, and HAF-Indonesia [HAF-Indo]) or (2) the stabilized HA stem with deletion of the variable head domain for less antigenic drift (e.g., © XXXX American Chemical Society

Received: January 7, 2019 Accepted: April 17, 2019

A

DOI: 10.1021/acs.analchem.9b00095 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

DE). Cell culture supernatant samples, vaccine reference materials, and null transfection media (cell culture media/ supernatant without vaccine product) were prepared in-house at the Vaccine Production Program Lab at the NIH (Gaithersburg, MD). Freestyle 293 expression media (used as the diluent for the VEE reference material) was purchased from Gibco (Grand Island, NY). Sample Preparation. The cell culture supernatant samples were tested without any preparation, except filtration through a 0.2 μm centrifuge membrane. Samples with estimated concentrations higher than the maximum linearity range were diluted and transferred into autosampler vials for analysis (50 μL loop injection size). Tandem-Column Setup for Separation. The IEC column and the SEC column were connected by a one-piece PEEK column coupler and installed onto the LC system in such a way that the mobile phase flowed through the IEC column and the one-piece PEEK column coupler into the SEC column, followed by UV detection at 215 nm. The separation was performed at ambient temperature. PBS (2×, pH 7.4) was used as mobile phase A, and 1 M NaCl was used as mobile phase B. Purified water (LC-MS grade) and 10% ethanol were used as mobile phases C and D, respectively. For H1ssF and HAF-Indo analyses, a step gradient was applied at a constant flow rate of 1 mL/min, with 100% mobile phase A flowing from 0−10 min. Afterward, the gradient transitioned to 100% mobile phase B (within 0.1 min) for an additional 10 min to strip the IEC column of the strongly bound charged species, followed by a quick step down to 100% mobile phase A (within 0.1 min) to re-equilibrate the IEC−SEC columns prior to the next injection. The total run time was 30 min per injection. For VEE analysis, a step gradient was also applied: 0−7 min (100% mobile phase A), 7−14 min (100% mobile phase B), and 14−21 min (100% mobile phase A), with a total run time of 21 min. After each sequence run, 100% mobile phase C was used to flush the system for at least 30 min, which was followed by a 10 min flush at 100% mobile phase D to properly store the columns before the system was shut down. For quantitation, peak areas were integrated automatically using the ApexTrack integration algorithm of the Empower software. Titer was determined by interpolating the peak area of each molecule from crude cell culture supernatant against the linear equation of the generated calibration curve.

frequently prevent affinity chromatography techniques from being practical methods for direct cell culture titer measurement. Other methods, such as immunoassays (e.g., ELISA, ECLIA, or biolayer interferometry), also pose challenges, such as long assay development times, high antibody costs, and poor robustness.13,14 Colorimetric or absorption protein assays (i.e., BCA, Bradford, A280, etc.) are not viable options either because of the necessity of a purified material.10 High-performance liquid chromatography (HPLC) coupled with UV detection is an attractive analytical tool for its selectivity, sensitivity, and reproducibility.15 Recently, an ion exchange chromatography (IEC) method was developed and used for total viral particle quantitation, whereas in a different case, a size exclusion chromatography (SEC) technique was applied for aggregate and content analysis of purified VLPs.16,17 A reversed-phase HPLC method has also been reported to quantify HA in nonpurified (in-process) samples, but a prepurification step was required.18 In this new method development, IEC was connected in tandem with SEC as a hyphenated chromatographic approach to separate the vaccine molecules from the rest of the components in cell culture media. Using this strategy, a strong anion IEC column was used to essentially act as a guard column and selectively retain the crude cell culture components, such as host cell proteins and other largely negatively charged species. Under the assay conditions, the vaccine particles, which were less negatively charged compared with the cell culture components, were assumed to not interact with the initial IEC column and pass directly through to the SEC column. As a second dimension, a hydrodynamic sizebased separation step was added to separate the large vaccine particles from any smaller species (of similar charge) in the cell culture media. This method was developed and applied successfully to all three types of vaccines (H1ssF, seasonal HAF, and encephalitic alphavirus VLPs) to measure product titer. In this technical note, development work is described with emphasis on H1ssF, whereas general application of the IEC−SEC method is exemplified using HAF-Indo and VEE vaccine candidates.



MATERIALS AND METHODS Chemicals and Instrumentation. All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), Thermo Scientific (Waltham, MA), or VWR (Radnor, PA) unless otherwise stated. For mobile phase preparation, LC-MS-grade water was purchased from Millipore (VWR), and 5 M sodium chloride (NaCl) and 10× phosphate-buffered saline (PBS, absent of calcium or magnesium) were purchased from Lonza (VWR). An ACQUITY H-Class liquid chromatography (LC) system and ultraviolet (UV) detector, both from Waters (Milford, MA), were used for the IEC−SEC assays. Empower 3 software (Waters) was used for data acquisition and processing. Different IEC columns (anion or cation exchangers) were evaluated during development, but the QSTAT AX strong anion exchange column (4.6 × 100 mm, 7 μm particles, nonporous), purchased from Tosoh Bioscience (King of Prussia, PA), was selected for the final method. The SRT SEC 300 column (7.8 × 150 mm, 5 μm particle size, 300 Å porosity), SRT SEC 500 column (7.8 × 150 mm, 5 μm particle size, 500 Å porosity), SRT SEC 1000 column (7.8 × 150 mm, 5 μm particle size, 1000 Å porosity), and a one-piece PEEK column coupler (to connect the IEC−SEC columns in series) were all purchased from Sepax Technologies (Newark,



RESULTS AND DISCUSSION SEC has been used to measure the concentrations of purified vaccine products; however, when applying SEC for crude cell culture H1ssF samples, coelution of interfering cell culture components with the vaccine product was observed. Figure 1 shows a representative UV profile of an H1ssF cell culture sample by SEC analysis. Incomplete separation was observed, indicating coexisting molecules of similar size to H1ssF (∼1 MDa in size). The large molecular weight species also accumulated as culture time increased. Therefore, adding another chromatography dimension was deemed necessary to fully resolve H1ssF for reliable quantitation. A strong anion exchange column was selected as a first dimension for the tandem IEC−SEC strategy to bind and delay any cell culture components with strong negative charge characteristics. It was hypothesized that the vaccine particle (theoretical pI of H1ssF protein: 5.4) was less negatively charged than the charged cell culture supernatant components, allowing the vaccine particle to elute through the IEC column within the B

DOI: 10.1021/acs.analchem.9b00095 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

strongly to the IEC column; this was followed by reequilibration using 2× PBS buffer. By successfully isolating the H1ssF vaccine particle in cell culture media via IEC−SEC, quantification was achievable for titer measurement using a calibration curve of purified H1ssF reference material. The combination of a strong anion IEC column with the SEC column was used for all three vaccine products. Figure 2 shows representative IEC−SEC chromatograms of the three vaccine particles (H1ssF, HAF-Indo, and VEE) in their corresponding transient transfection cell culture media. Overall, because similar cell culture processes were applied for all these vaccine developments, the same strong anion IEC column and mobile phases were used. However, it should be noted that if different types of cell lines are used, different IEC modes may be needed. For SEC separation, exclusion of the vaccine particle in the pore network allowed better separation of the vaccine particles from the cell culture species. To this end, the vaccine particles eluted within the void volume (flowthrough fraction, retention time: ∼4 min) as a single peak, which enabled simple peak integration and subsequent quantification. For example, an SEC column with a 300 Å pore size was employed for separation of the VEE VLP, which had a particle size of ∼70 nm (by electron microscopy). This strategy allowed for separation of the VLP (as a single peak) from the cell culture media with good resolution and was suitable for routine and straightforward quantification. Furthermore, utilizing an SEC column with a 300 Å pore size yielded good separation of H1ssF (∼25 nm) and HAFIndo (∼35 nm) from the cell culture supernatant for subsequent quantification of a single peak. In addition to reaching the goal of titer quantification, the method was further refined to be able to monitor both titer and percent

Figure 1. Representative chromatogram of H1ssF coeluting with cell culture species when SEC only was applied for separation.

void volume and directly proceed to the SEC column. Using 2× PBS (with ∼300 mM NaCl) as the loading buffer limited H1ssF from interacting with the IEC stationary phase, while the cell culture debris and components with more negative charge were retained. Relatively weak binding of H1ssF to the ion exchange column was observed when lower salt concentrations (1× or 1.5× PBS) were used for mobile phase A. Furthermore, the vaccine particles (H1ssF, HAFIndo, and VEE) had diameter sizes between 20 and 80 nm, whereas most host cell proteins and other cell culture components were smaller. Therefore, SEC as the second dimension of chromatography, was able to sieve the remaining particles and proteins (of similar charge) by size, thereby reaching the goal of complete separation for H1ssF. After H1ssF and some of the cell culture components were eluted and separated, the columns were rejuvenated with 1 M salt to further release any negatively charged species that bound

Figure 2. Representative IEC−SEC chromatograms of (a) H1ssF (SEC pore size: 500 Å), (b) HAF-Indo (SEC pore size: 1000 Å), and (c) VEE (SEC pore size: 300 Å) showing each analyte of interest separated from the cell culture components. The same IEC column was coupled in tandem with an SEC column, but different SEC pore sizes were utilized depending on the molecule. C

DOI: 10.1021/acs.analchem.9b00095 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

H1ssF) eluted at a later time. Figure 3d shows the profile of H1ssF (final: 100 μg/mL) spiked into the null transfection media. The method was deemed specific; the H1ssF peak was well-resolved from the rest of the peaks. Good specificity for VEE and HAF-Indo in their corresponding cell culture media was also observed using this IEC−SEC strategy (Figure 2). The LOQ for H1ssF in 2× PBS was identified to be 15 μg/ mL (S/N > 20). The simple criterion of 85−115% spike recovery was also achieved when H1ssF (at 15 μg/mL or 9.0 × 1012 particles/mL) was spiked into null transfection media (without H1ssF). The LOQs for HAF-Indo and VEE were both determined to be 10 μg/mL using a similar approach. Linearity of the method was also assessed, and the correlation coefficient (R2) values were greater than 0.997 for each vaccine product. In summary, calibration curves were typically generated in the following concentration ranges: 15−300 μg/ mL for H1ssF (R2 > 0.999), 10−300 μg/mL for HAF-Indo (R2 > 0.999), and 10−200 μg/mL for VEE (R2 > 0.997, see Figure S2 in the Supporting Information). Because minimal matrix effects were observed (i.e., minimal difference between the slopes of the curves when using 2× PBS vs the null transfection media), 2× PBS was chosen to be used as the diluent for H1ssF and HAF-Indo for the calibration curves. For the VEE VLP vaccine product, however, null transfection (or commercial cell culture media solution) was required as the diluent to normalize matrix effects. The method accuracy was also demonstrated by spike recovery experiments: H1ssF was spiked into either 2× PBS or null transfection media with targeted concentrations of 25, 50, and 100 μg/mL. As summarized in Table 1, percent recovery of H1ssF was between 90 and 110%, showing good method accuracy with minimal matrix interference. Assay precision was also verified by preparing H1ssF (final: 100 μg/mL) in null transfection media followed by analysis using two different LC instruments with different columns and buffers prepared on different days. The average measurement of H1ssF among the runs was 93% of the target value with a relative standard deviation (CV) of 1.7% (n = 12), demonstrating good precision (see Table S1 in the Supporting Information). In summary, this method has been successfully applied for routine titer measurements. Figure 4 shows an example of H1ssF titer measurements in cell culture media from day 2 (33.9 μg/mL) up to day 5 (144 μg/mL). As demonstrated, from method development and quality assessment to application, this IEC−SEC method was capable of monitoring titer directly in cell culture supernatant to provide timely feedback for cell culture development. Up to 1000 injections were achieved for the column lifetime. The assay was also further applied to other vaccine candidates, such as the

aggregate for the influenza products (H1ssF and HAF-Indo) in cell culture by employing an SEC column with a larger pore size (e.g., 500 or 1000 Å; see Figure S1 in the Supporting Information). Good resolution between the vaccine molecule and cell culture components was maintained, and as an additional benefit, aggregate from monomer could be resolved when utilizing the larger SEC pore size (inclusion of the pore network). Although the primary goal of this work was to achieve vaccine titer measurement, the aggregate information was an added value for process monitoring, and both aggregate and monomer contributed towards titer determination. Method specificity was demonstrated in Figure 3 for H1ssF under its optimized parameters. As shown in Figure 3a, no

Figure 3. Stacked chromatograms of (a) 2× PBS only, (b) H1ssF (100 μg/mL) spiked into 2× PBS, (c) null transfection media only (cell culture without H1ssF, negative control), and (d) H1ssF (100 μg/mL) spiked into null transfection media.

peaks were observed for the 2× PBS buffer (blank) injection. Figure 3b exhibits a distinct peak observed around 4 min for H1ssF when purified H1ssF (final: 100 μg/mL) was spiked into 2× PBS. Figure 3c is the profile of the null transfection media, indicating that all cell media components (without Table 1. Accuracy Assessment of the IEC−SEC Methoda

H1ssF spiked into 2× PBS

H1ssF spiked into null transfection media

target concentration (μg/mL)

concentration (μg/mL)

recovery (%)

concentration (μg/mL)

recovery (%)

15 25 50 100 200 300

15.6 27.1 51.4 100.9 197.6 301.6

104.0 108.4 102.8 100.9 98.8 100.5

NA 24.8 46.3 93.3 NA NA

NA 99.2 92.6 93.3 NA NA

a

Average percent recovery of H1ssF was determined by recovery of spiked, purified H1ssF into either 2× PBS or null transfection media. Reported values were determined by interpolating against an external calibration curve of H1ssF prepared in 2× PBS buffer. D

DOI: 10.1021/acs.analchem.9b00095 Anal. Chem. XXXX, XXX, XXX−XXX

Technical Note

Analytical Chemistry

helpful review and input; and Jessica Bahorich and Kevin Carlton for project support. This work was supported by the Intramural Research Program of the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH).



Figure 4. Representative chromatographic overlays showing H1ssF cell culture growth measurement starting from day 2 through day 5.

encephalitic alphavirus VLPs and seasonal HAF nanoparticles in their cell culture developments, demonstrating its broader applicability as a new platform IEC−SEC approach to support upstream titer testing with a significantly improved turnaround time.



CONCLUSION To our knowledge, this is the first described IEC−SEC tandem chromatography method to support fast turn-around titer measurement of nanoparticle and VLP vaccine products in cell culture media, allowing direct monitoring of vaccine molecule growth in highly complex cell culture media without the need of a prior purification step. The method is sensitive, accurate, and specific and can deliver reliable titer measurements quickly during cell culture development. As such, this new quantitative approach was successfully applied to support multiple nanoparticle and VLP vaccine programs.



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b00095. Screening SEC columns with different pore sizes for IEC−SEC method development, representative calibration curves of the various vaccine particles, and intraand intermediate precision assessment of the IEC−SEC assay for H1ssF (PDF)



REFERENCES

(1) Settembre, E. C.; Dormitzer, P. R.; Rappuoli, R. Hum. Vaccines Immunother. 2014, 10, 600−604. (2) Poland, G. A.; Rottinghaus, S. T.; Jacobson, R. M. Vaccine 2001, 19, 2216−2220. (3) Wolfe, D. N.; Heppner, D. G.; Gardner, S. N.; Jaing, C.; Dupuy, L. C.; Schmaljohn, C. S.; Carlton, K. Am. J. Trop. Med. Hyg. 2014, 91, 442−450. (4) Yassine, H. M.; Boyington, J. C.; McTamney, P. M.; Wei, C. J.; Kanekiyo, M.; Kong, W. P.; Gallagher, J. R.; Wang, L.; Zhang, Y.; Joyce, M. G.; Lingwood, D.; Moin, S. M.; Andersen, H.; Okuno, Y.; Rao, S. S.; Harris, A. K.; Kwong, P. D.; Mascola, J. R.; Nabel, G. J.; Graham, B. S. Nat. Med. 2015, 21, 1065−1070. (5) Kanekiyo, M.; Wei, C. J.; Yassine, H. M.; McTamney, P. M.; Boyington, J. C.; Whittle, J. R.; Rao, S. S.; Kong, W. P.; Wang, L.; Nabel, G. J. Nature 2013, 499, 102−106. (6) Impagliazzo, A.; Milder, F.; Kuipers, H.; Wagner, M. V.; Zhu, X.; Hoffman, R. M. B.; van Meersbergen, R.; Huizingh, J.; Wanningen, P.; Verspuij, J.; de Man, M.; Ding, Z.; Apetri, A.; Kükrer, B.; SneekesVriese, E.; Tomkiewicz, D.; Laursen, N. S.; Lee, P. S.; Zakrzewska, A.; Dekking, L.; et al. Science 2015, 349, 1301−1306. (7) Thompson, C. M.; Petiot, E.; Mullick, A.; Aucoin, M. G.; Henry, O.; Kamen, A. A. BMC Biotechnol. 2015, 15, 31. (8) Carvalho, S. B.; Moleirinho, M. G.; Wheatley, D.; Welsh, J.; Gantier, R.; Alves, P. M.; Peixoto, C.; Carrondo, M. J. T. Biotechnol. J. 2017, 12, 1700031. (9) Toprani, V. M.; Cheng, Y.; Wahome, N.; Khasa, H.; Kueltzo, L. A.; Schwartz, R. M.; Middaugh, C. R.; Joshi, S. B.; Volkin, D. B. J. Pharm. Sci. 2018, 107, 2544−2558. (10) Gollapudi, D.; Wycuff, D. L.; Schwartz, R. M.; Cooper, J. W.; Cheng, K. Electrophoresis 2017, 38, 2610−2621. (11) Xu, D.; Marchionni, K.; Hu, Y.; Zhang, W.; Sosic, Z. J. Pharm. Biomed. Anal. 2017, 145, 10−15. (12) Thompson, C. M.; Petiot, E.; Lennaertz, A.; Henry, O.; Kamen, A. A. Virol. J. 2013, 10, 141−141. (13) Vajda, J.; Weber, D.; Brekel, D.; Hundt, B.; Müller, E. J. Chromatogr. A 2016, 1465, 117−125. (14) Baker, K. N.; Rendall, M. H.; Patel, A.; Boyd, P.; Hoare, M.; Freedman, R. B.; James, D. C. Trends Biotechnol. 2002, 20, 149−156. (15) Shytuhina, A.; Pristatsky, P.; He, J.; Casimiro, D. R.; Schwartz, R. M.; Hoang, V. M.; Ha, S. J. Chromatogr. A 2014, 1364, 192−197. (16) Transfiguracion, J.; Manceur, A. P.; Petiot, E.; Thompson, C. M.; Kamen, A. A. Vaccine 2015, 33, 78−84. (17) Ladd Effio, C.; Oelmeier, S. A.; Hubbuch, J. Vaccine 2016, 34, 1259−1267. (18) Kapteyn, J. C.; Saidi, M. D.; Dijkstra, R.; Kars, C.; Tjon, J. C. M. S. K.; Weverling, G. J.; de Vocht, M. L.; Kompier, R.; van Montfort, B. A.; Guichoux, J.-Y.; Goudsmit, J.; Lagerwerf, F. M. Vaccine 2006, 24, 3137−3144.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: 1-301-761-7288 (Q.P.L.). *E-mail: [email protected]. Tel.: 1-301-761-7574 (N.A.S.). ORCID

Q. Paula Lei: 0000-0002-3656-4014 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors thank Lori Romaine, Gengcheng Yang, Nathan Barefoot, Alison Vinitsky, Yanhong Yang, Mandy Alger, and Fan Yang for their scientific input. The authors also acknowledge Lisa Kueltzo, KC Cheng, Dan Gowetski, and Adam Charlton for their helpful discussions; Joe Horwitz’s E

DOI: 10.1021/acs.analchem.9b00095 Anal. Chem. XXXX, XXX, XXX−XXX