CVA-MS Analysis To Investigate The Effects Of Leachables From Single-Use Bioreactors On

Monoclonal Antibodies

Sara Carillo1, Florian Füssl1, Noemí Dorival Garcia1, Christine Ta1, Paul S Kelly1, Niall Barron1,2, Jonathan Bones1,2

1Characterisation and Comparability Laboratory, NIBRT. Foster Avenue, Mount Merrion, Blackrock, co. Dublin. Ireland2School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4

Background

The last decade saw exponential growth of biopharmaceuticals. Biomanufacturing is evolvingtogether with the need to reduce capital costs and increase flexibility and versatility ofproduction sites. To achieve these goals, the implementation of single-use devices for industrialscale bioproduction is a key objective. A drawback of the introduction of these disposablepolymeric materials is that many of these produce breakdown products that could interferewith cell culture in the upstream process or pose a risk to the drug product during downstreamand fill/finish operations.1-4

This research focuses on the cellular response of Chinese hamster ovary (CHO) cell linesfollowing exposure to extractables and leachables from single-use bioreactors using an anti-IL8IgG1 producing cell line, CHO DP-12. Cells were cultured using media pre-conditioned in twosingle-use bioreactors with different leachables profiles under normal operation conditions.The consequences of leachable exposure on mAb1 product quality attributes (PQAs) have beenevaluated using several orthogonal techniques.Charge variant analysis hyphenated with mass spectrometry (CVA-MS)5,6 was found to behighly informative and enabled the characterisation of differences between the two productsthat were not identified with other techniques. We used CVA-MS to characterise anti-IL8 IgG1produced by CHO-DP12 in its native conformation and we were able to pinpoint posttranslational modifications (PTMs) induced by the exposure to the leachables cocktail with highmass accuracy for a confident identification of the proteoforms.

Toxicology

Conditioned media was prepared in triplicate for each film tested along with a negative control incubated inglass Duran bottle. Media from each bag/bottle was used to prepare biological triplicates of CHO-DP12 cellculture and Fresh BalanCD media (Irvine Scientific) was used as an additional negative control. Cells werecultured in 250 mL flasks. Viable cell density (VCD) and cellular viability was assessed using a benchtop flowcytometer, Guava EasyCyte 5HT system (Millipore, Billerica, MA) in combination with the ViaCount assay(Millipore) as per manufacturer’s specifications. IgG titre was obtained from ELISA analysis on cell supernatant.For IgG analysis only cells cultured in media preconditioned in Film 1 were used.

ModificationConditioned

Media (Rel%)Control (Rel%)

M4+Oxidation 1.54 1.09N163+Deamidation 0.34 0.37N55+Deamidation 3.44 3.90D73+Isomerization 8.85 6.99N77+Deamidation 0.49 0.51

M83+Oxidation 0.90 1.11N84+Deamidation 0.79 0.83M257+Oxidation 8.06 6.17

W282+Double Oxidation 0.12 0.17D285+Isomerization 0.69 1.11N291+Deamidation 0.27 0.34

N302+A1G0F 7.90 9.13N302+A1G1F 2.30 3.09N302+A2G0 1.87 1.44

N302+A2G0F 48.26 42.32N302+A2G1 0.69 0.60

N302+A2G1F 33.21 35.94N302+A2G2F 3.73 4.99

N302+M5 0.57 0.79N302+Unglycosylated 1.38 1.55

W318+Double Oxidation 2.25 1.59N320+Deamidation 1.54 2.20

K331+Glycation 0.42 0.24M363+Oxidation 0.59 0.65

N366+Deamidation 1.15 1.28~N389+Deamidation 0.88 1.36D406+Isomerization 0.71 0.69

G451+Lys 2.72 3.52

PQAs screening on Film-1 anti-IL8 IgG1

Peptide mapping The analysis was performed on technical replicates of 3 biological replicates for triplicate conditioned media preparation (n=27). Samples were digested using King Fisher Duo automated platform with SMART Digest™ trypsin magnetic kit. Tryptic digests were analysed via LC-MS/MS analysis on a Vanquish Acclaim C18 2.1 x 250mm column interfaced with a Q Exactive™ Hybrid quadrupole Orbitrap Mass Spectrometer. Data analysis was performed on BioPharma Finder™ 3.0 software (Confidence Score>95). Mass tolerance 0.5ppm

N-glycans were released with PNGase F (CarboClip), 2-AB labelled and analysed with LC-MS using a Accucore™ Amide HILIC 2.1 x 150mm column (Thermo Scientific) and a gradient of 50 mMammonium formate pH 4.4 and acetonitrile for separation. Mass Spec analysis was performed on a Q ExactivePlus hybrid quadrupole Orbitrap mass spectrometer in negative mode.

Aggregate analysis was performed via LC-UV analysis on a Vanquish Flex UHPLC (Thermo Fisher) equipped with a MAbPac™ SEC-1 4.6 x 300mm column in isocratic mode using isocratic conditions of 50 mM sodium phosphate buffer, 300 mM NaCl, pH 6.8. Triplicate injections for each biological replicate were performed (n=27)

2AB N-GLYCANGLASSFILM 1

SEC-UV Aggregate analysisGLASSFILM 1Blank

CVA-MS MATERIALS & METHODSCharge variant analysis was performed on pooled

biological replicates (n=9) for control samples grown in

media preconditioned in glass bottles and conditionedmedia from bioreactor F-1. 20 µg of each purified mAb

were injected in triplicate on a Vanquish Flex uHPLC usinga MAbPac SCX-10 RS 2.1 x 50 mm, flow rate of 0.4 mL/min,using mobile phases reported below and the gradient asillustrated in Table 1.LC was hyphenated with a Q Exactive™ Plus hybridquadrupole Orbitrap mass spectrometer, using thefollowing settings: scan range 2,500-8,000 m/z, resolution35,000, microscan 10, Capillary temp 275°C, S-lens 200,spray voltage 3.6kV, sheath gas 20, aux gas 10. HMR modeon.Deconvolution was performed using BioPharma FInder™v3.0 software, using Sliding Window option. Min adjacentcharge state 4, scan offset 1%, sliding window mergetolerance 20 ppm. All identified masses were obtainedwith mass accuracies higher than 25 ppm.

Table1: CVA-MS Gradient condition

Time (min)

%B Curve

0.0 50 5

2.0 85 5

10.0 100 3

10.1 100 5

12.0 100 5

12.1 0 5

14.0 0 5

14.1 50 5

25.0 50 5

Mobile phase A: 25 mMammonium bicarbonate, 30 mM acetic acid. pH 5.3Mobile phase B: 10mM ammonium hydroxide, final pH 10.9

Following deconvolution, no species were assigned to the peak present in conditioned media sample at RT 6.7min. The presence of an additional charge envelope between 6,800-8,000 m/z lead to the hypothesis that the peakwas a dimer.Deconvolution performed on the highlighted peak considering an extended output mass range returned signalscorresponding to twice the mass of our anti-IL8 IgG1 (Δppm=36) and showing glycosylation differences around 162Da. This peak is not present in control sample.

Single-use technology is representing a significant change in bioprocess design and the consequences of the introduction of these plastic materials needs to be closely monitoring to assess anydeviation in product quality profiles, to ensure efficacy and patient safety. Preconditioning cell culture media within two types of single-use bioreactors showed dramatic differences in performance.Toxicology and cell growth analysis proved F-1 to modify the media causing a detrimental effect on cell growth and associated impact on IgG titre, compared to media incubated under normaloperating conditions (7 days, 37°C). A second bioreactor showed no difference between cells grown and IgG produced in preconditioned media with respect to the control (media preconditioned inglass)7.Preliminary analysis on the sample produced from cells growing in the media preconditioned in F-1 showed negligible difference in mAb PQAs; peptide mapping showed a small increase in oxidationlevels with an overall increase of 3%, N-glycan analysis showed a decrease in terminal galactosylation (data confirmed via peptide mapping) and size exclusion LC-UV analysis did not reveal anydramatic change in aggregation. CVA-MS was performed on the two samples, showing for the first time marked differences in the charge variants profile between the two samples. Mediapreconditioned in F-1 appears to induce C-terminus modification consisting in the complete deletion of terminal lysine and subsequent deletion of C-terminal glycine leading to terminal prolineamidation on one or both heavy chains (total Δ mass= -58.029/-116.058 Da). The biological consequences of proline amidation have not been widely reported in the literature8. CVA-MS also revealedevidence of non-complete cleavage of the signal peptide on the N-terminus, being present at the same level in both samples. These modifications were confirmed by peptide mapping after modifyingthe data processing method. CVA-MS also allowed the separation of a dimeric species that was not observed by SEC-UV analysis.

While some concerns still remain on the consequences of single-use technologies on product quality attributes, it is pivotal to improve analytical technologies to allow accurate monitoring ofbiotherapeutic PQAs. CVA-MS and native MS proved to be highly informative with no sample preparation and to generate highly accurate mass spec data. Moreover, in this circumstance, CVA-MS hasbeen able to highlight features that were not observed with other techniques.

References1. Hammond M. et al., PDA J. Pharm. and Tech., 2013 (67), 123-134.

2. Hammond M. et al., Biotechnol. Prog., 2014 (30), 332-337.3. Kelly P.S. et al. Biotechnol. Prog., 2016 (32), 1547-1558.

4. Dorival-Garcia et al. Anal. Chem. 2018, 90(15), 9006-90155. Fussl et al. Anal. Chem. 2018, 90(7), 4669-46766. Fussl et al mAbs 2019, 11(1), 116-1287. Kelly P.S. et al. Biotechnol. Prog. Submitted8. Kaschak T et al. mAbs 2011, 3(6), 577-583.

CVA-MS Control

Conditioned media

*Only G0F_G1F glycoformare marked. For the main peak all N-glycan variants are marked with a smaller diamond

Results and Discussion

Conclusions