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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Anal Biochem. 2021 Oct 20;634:114425. doi: 10.1016/j.ab.2021.114425

Analysis of N15-rat growth hormone after incubation with rat subcutaneous tissue and immune cells using ultra-pressure chromatography-mass spectrometry

Ninad Varkhede 1,2, Björn-Hendrik Peters 1,3, K Ryan Moulder 1, Philip Gao 4, Christian Schöneich 1, M Laird Forrest 1,5
PMCID: PMC8599641  NIHMSID: NIHMS1750905  PMID: 34678250

Abstract

Therapeutic proteins (TPs) are exposed to various immune cells like macrophages and neutrophils, especially after subcutaneous (SC) administration. It is well known that the immune cells can generate reactive oxygen species (ROS) and this may lead to oxidation of TPs. The oxidation can occur in the SC tissue after SC administration, during distribution to the immune organs like lymph nodes and spleen, and even in the blood circulation. The oxidation can lead to alteration of their pharmacokinetics and efficacy. Therefore, it is important to study the oxidation of TPs in the biological matrices using ultra-pressure chromatography-mass spectrometry. Rat growth hormone (rGH) was selected as a test protein due to its similarity with human growth hormone (hGH), which is widely used for treatment of growth hormone deficiency. In this manuscript, we have summarized sample processing strategy and ultra-pressure chromatography-mass spectrometry methodology to identify rGH and its degradation products after ex-vivo incubation with rat SC tissue, and in vitro incubation with rat splenocytes and canine peripheral blood mononuclear cells (cPBMCs) as a model foreign host species. We did not observe oxidation of rGH in these biological matrices. This could be due to very minor yields of oxidation products, lack of sensitivity of the mass spectrometry method, loss of protein during sample processing, rapid turnover of oxidized protein or a combination of all factors.

Graphical Abstract

graphic file with name nihms-1750905-f0001.jpg

1. Introduction

Oxidation of therapeutic proteins (TPs) can lead to altered pharmacokinetics, reduced biological activity and increased immunogenicity [1–3]. Amino acids like methionine, tryptophan, tyrosine, phenylalanine, histidine, and cysteine are commonly oxidized by reactive oxygen species (ROS). After subcutaneous (SC) administration, TPs are often exposed to various immune cells in the skin, SC tissue and the lymphatic system [3]. These immune cells are known to generate ROS [3–5]. We selected rat growth hormone (rGH) as a test protein for adventitious oxidation during incubation with rat SC tissue, rat splenocytes and canine peripheral blood mononuclear cells (cPBMCs) as rGH and human growth hormone (hGH) have considerable amino acid sequence homology [6]. In addition, hGH is administered subcutaneously for therapeutic purposes [7]. It is commonly used to treat growth hormone deficiency [8, 9]. hGH is used for chronic administration over a period of many years. Therefore, the generation of anti-hGH antibodies against native, misfolded or chemically modified protein is of concern. For example, hGH antibodies were found in pediatric studies in 75% patients after 12 months of treatment [10], and in another report, loss of hGH efficacy was observed despite dose escalation in a child with a high titer of anti-hGH antibodies [2, 11].

The SC tissue is filled with many immune cells like macrophages which are known to generate ROS [3]. Therefore, ex-vivo incubation of N15-rGH with rat SC tissue was performed. In this study, N15labeled rGH (N15-rGH) was used for incubation with the rat SC tissue to distinguish it from potential endogenous rGH. The use of N15-rGH would help to distinguish the peptides in the tryptic digest from any potential peptides originating from the endogenous rGH present in the SC tissue. In addition, splenocytes [12] and PBMCs [13, 14] may also generate ROS. Therefore, splenocytes and PBMCs were used for in vitro incubation with N15-rGH. The hypothesis was that ROS generated by the immune cells can oxidize N15-rGH. Further, to enhance production of ROS, lipopolysaccharide (LPS) was injected at the SC tissue site in rats, and the SC tissue was excised afterwards [15, 16]. LPS was also added at appropriate concentration to the splenocyte and cPBMC cultures to activate the cells for increased production of ROS [17–20].

Interference due to the peptides originating from the SC tissue and immune cell cultures was a major challenge in the analysis of N15-rGH. The ultra-pressure chromatography was used to separate various peptides for further detection by mass spectrometry. This study demonstrated that rGH can be identified after ex-vivo and in vitro incubations in the biological matrices like SC tissue, and immune cell cultures. The methodology can be applied to other TPs to study oxidation due to ROS generated by the immune cells.

2. Materials and Methods

2.1. Materials

The 2,2’-azobis (2-methylpropionamidine) dihydrochloride (AAPH), potassium ferricyanide, 1,10-phenanthroline monohydrate, sodium citrate tribasic dihydrate, and ascorbic acid were purchased from Sigma Aldrich (St. Louis, MO). Ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), LC/MS-grade water (0.1% v/v formic acid), acetonitrile (0.1% v/v formic acid), monobasic sodium dihydrogen phosphate, sulfuric acid, hydrogen fluoride, dibasic sodium hydrogen phosphate, and the Pierce BCA protein assay kit (Product number: 23227) were purchased from Fisher Scientific (Waltham, MA). Trypsin from porcine pancreas (Sigma Aldrich, St. Louise, MO) was used for tryptic digestion of the samples. Amicon ultra 0.5 centrifugal filter devices (10, 30, 100 kDa cut off membranes) were purchased from Millipore Inc., (Bedford, MA, USA).

2.2. Production of N15-rat growth hormone (N15-rGH)

Production of rGH was described in our previous publication related to interaction of the protein with iron oxide nanoparticles. The method described below is quoted from the same publication [2]. However, few modifications are included for N15 isotope labeling of rGH. rGH is a 22 kDa protein of 191 amino acids that contains two disulfide bonds [6, 21]. The cDNA plasmid for the rGH protein sequence (Protein accession number: AAI66872) was synthesized by GenScript (New Jersey, USA) and inserted into pET-28a(+) using the Ndel and BamHI restriction sites [22]. The pre-protein sequence (first 25 amino acids) was removed from the original cDNA for production in E. coli.; the resulting rGH amino acid sequence is shown in the Supplementary Figure S1. The plasmid was transformed into DH5α competent E. coli cells, and a single colony was expanded, mini-prepped, and the sequence confirmed by KanPro Research Inc. (Lawrence, KS).

The confirmed rGH plasmid was transformed into BL21 (DE3) E. coli, and rGH was produced by shake flask culture using a protocol modified from previously reported methods [21, 23, 24]. In brief, the E. coli cells were cultured in lysogeny broth (LB) media with kanamycin (40 μg/mL) and chloramphenicol (40 μg/mL) at 37 °C and 200 rpm shaking. Once an optical density (at 600 nm) of ca. 0.6 was reached, cells were pelleted at 1391×g for 10 min, the pellet was washed with and then re-suspended in M9 media without ammonium chloride. The re-suspended cells were incubated for 1 h. After the 1 h incubation, 1 g ammonium-15N chloride was added to the culture. The culture was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) (1.0 mM) and incubated. After 4 h, cultures were centrifuged at 1391×g for 10 min and the cell pellet was suspended in lysis buffer (50 mM tris-hydrochloride, 500 mM sodium chloride, pH 8). The mixture was then sonicated (50% power, 4 cycles × 30 sec, at 4 °C) until it was no longer viscous using a Fisher Scientific Sonic Dismembrator Model 500 (Waltham, MA). Inclusion bodies of rGH were isolated by centrifuging at 6,000×g (at 4 °C) for 30 min, followed by a water wash, and then dissolved in 6 M urea lysis buffer. The proteins were refolded by following proprietary procedures (KanPro Research Inc.), and then dialyzed in phosphate buffer saline (PBS), containing 88 mM of mannitol. N15-rGH was analyzed for purity using SDS-PAGE (Supplementary Figure S2). Detailed analysis of a tryptic digest of the protein (sequence coverage of ~56%) using mass spectrometry was completed. The rGH proteins was digested using trypsin and peptide fragments were analyzed using mass spectrometry in previous studies [2, 23]. Those studies were used as a reference to analyze peptide fragments of N15-rGH after tryptic digestions.

2.3. Animals

Male Fisher rats were purchased from Charles River (Wilmington, MA). The animals were acclimatized for 3 days and housed as 2 animals per cage or individually if a cage mate was not available. Animals were approximately 21–56 days old at the start of the study. A 12 h light and 12 h dark cycle was maintained throughout the study. Rats were fed and given access to water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kansas. Whenever possible, procedures were designed to avoid or minimize discomfort, distress, and pain to the animals.

2.4. Preparation of rat splenocyte suspension

Gey’s lysis solution was prepared fresh by mixing stock A (20%), stock B (5%), stock C (5%) and autoclaved water (70%). Stock A contained ammonium chloride (654 mM), potassium chloride (24.8 mM), disodium hydrogen phosphate (4 mM), potassium dihydrogen phosphate (8.8 mM), glucose (27 mM), and phenol red (50 mg/1000 mL). Stock B contained magnesium chloride hexahydrate (20 mM), magnesium sulfate heptahydrate (5.6 mM), and calcium chloride (30 mM). Stock C was 267 mM sodium bicarbonate solution. Stocks A, B and C were freshly prepared in water and autoclaved. Sterile conditions were maintained while mixing the stocks to make the Gey’s lysis solution.

The rat splenocytes were cultured according to a previously published protocol [25]. The rats were euthanized by exsanguination and the spleen was removed. The spleen was immediately transferred to a 50 mL Falcon tube containing 5 mL of sterile RPMI media with 1% Penicillin-Streptomycin (Pen/Strep). This mixture was then transferred to a small petri dish containing a wire mesh in sterile condition. The spleen was mashed on the wire mesh using the rubber end of a 1 mL sterile syringe plunger. The larger cell debris was removed, and the cell suspension was transferred to a 15 mL Falcon tube and centrifuged at 2650 × g for 4.5 min. The cell pellet was re-suspended in 1 mL RPMI media containing 10% fetal bovine serum (FBS) and 1% P/S, and 3.5 mL of 1× Gey’s lysis solution was added to the re-suspended cell pellet and incubated on ice for 3.5 min. Then, 10 mL of RPMI (10% FBS) was added, and the cells were centrifuged at 2650 × g to get a pellet. The media was decanted, and the cell pellet was re-suspended in 10 mL of RPMI media (10% FBS and 1% Pen/Strep). The cells were counted using a Luna-II Automated Cell Counter (Logos Biosystems, South Korea). The cells were plated in a 12 well plate at 20 million cells/well in 1 mL media and incubated with rGH.

2.5. Preparation of canine peripheral blood mononuclear cell (cPBMC) suspension

A previously published protocol was used to prepare cPBMC culture [26, 27]. Blood from healthy dog donors (age 7–10 years) was obtained with appropriate informed consent of the owner and under approved institutional guidelines. Blood (8 ml per tube) was drawn via sterile venipuncture into a BD Vacutainer cell preparation tube containing sodium heparin (BD Biosciences, San Jose, CA). cPBMCs were isolated within 1 h following procurement by density gradient centrifugation according to the manufacturer (BD Biosciences) protocol and re-suspended to 1×106 cells/mL in sterile RPMI 1640 medium with L-glutamine (ThermoFisher Scientific, Waltham, MA) supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin (Corning, Corning, NY) and 10% (v/v) heat-inactivated Fetalgro (RMBIO, Missoula, MT). The isolated cPBMCs were used for incubation with N15-rGH.

2.6. Incubation of N15-rGH with splenocytes and cPBMCs

rGH (0.5 mg/mL) was incubated with the spleen cell suspension or cPBMCs with cell count of 1×107 cells/mL or 1×106 cells/mL, respectively. LPS was added to both rat splenocytes (10 μg/mL) and cPBMCs (2 μg/mL) [17–20]. The cells were incubated at 4 or 37 °C for 3, 12, and 24 h and a 0 h sample was collected as a control. At the given time point, samples were immediately stored at −80 °C until the further processing described in Sections 2.8 and 2.9.

2.7. Ex vivo incubation of N15-rGH with rat SC tissue

The hair in the back region of rat were removed using trimmer. LPS (1 mL of 1 mg/mL, 4 mg/kg) was administered by SC route on the back of rat to enhance ROS production [15, 16]. LPS was retained at the SC injection site after administration for several weeks according to a published study with radiolabeled LPS [28]. Therefore, it can be assumed that LPS was available for longer duration to enhance ROS production. After 24 h, the animal was euthanized by exsanguination. The SC tissue of around 10 mm2 was collected. The tissue was incubated with N15-rGH (1 mg) at 37 °C for 2 h. The supernatant was removed from the incubation. In addition, the SC tissue was washed using water to remove any traces of N15-rGH. The collected sample was processed as described in sections 2.8 and 2.9.

2.8. Reduction and alkylation of N15-rGH

The N15-rGH incubated with rat SC tissue, splenocytes or cPBMCs was processed further by reduction of disulfide bonds using dithiothreitol, and alkylation of cysteine residues using iodoacetamide to prevent reformation of disulfide bonds. The disulfide bonds were reduced to increase efficiency of tryptic digestion by disrupting secondary structure of the protein. To a sample, 50 μL of dithiothreitol (100 mM stock solution in 50 μM ammonium bicarbonate buffer) was added to obtain a final concentration of 5 mM. The mixture was incubated at 55 °C for 1 h. The reduced cysteine residues were alkylated by adding 80 μL of iodoacetamide (200 mM stock solution in 50 mM ammonium bicarbonate buffer) to obtain a final concentration of 15 mM. The mixture was incubated at 37 °C for 1 h.

2.9. Tryptic digestion of N15-rGH

The reduced and alkylated N15-rGH was precipitated with 800 μL of 0.5 M perchloric acid. The samples were centrifuged at 1500×g for 15 min, and the resulting pellet was suspended in 400 μL of 50 mM ammonium bicarbonate buffer. Trypsin from porcine pancreas (0.25 mg, Sigma Aldrich, St. Louise, MO) was added and the samples were incubated overnight at 37 °C. The proteolytic digests were centrifuged using Amicon ultra 0.5 centrifuge filters (10 kDa) to remove trace amounts of undigested N15-rGH and trypsin. The tryptic peptides were stored at −20 °C until the mass spectrometry analysis.

2.10. Ultra-pressure chromatography-mass spectrometry analysis

A nanoAcquity UPLC (Waters Corporation, Milford, MA) connected to a Xevo Q-TOF (Waters Corporation, UK) was used for LC-ESI-MS experiments. Tryptic peptides were first trapped on an UPLC Symmetry C18 nanoAcquity trap column (5 μm, 180 μm × 20mm, Water Corporation) and then separated on an UPLC Peptide CSH C18 nanoAcquity column (1.7 μm, 75 μm x 250 mm, Waters Corporation). For separation and analysis, we applied an optimized method and previously reported mass spectrometry parameters [2, 29]. In silico tryptic digestions of rGH and prediction of fragment ions were performed with the web-based application, Proteomics Toolkit (Institute of Systems Biology, Seattle, WA) and ProteinProspector 5.21.2 (The University of California, San Francisco, CA).

3. Results

3.1. Separation and identification of N15-rGH peptides using ultra-pressure chromatography-mass spectrometry after tryptic digestion

56% of N15-rGH peptides were identified using mass fragmentation pattern obtained after tryptic digestion and mass spectrometry analysis of naive N15-rGH. Supplementary Table S1 has tabulated a list of peptides which were not recovered or detected in the analysis. Supplementary Figures 4 to 15 demonstrate identification of the peptides originating from rGH using mass fragmentation pattern.

Supplementary Figure S3 shows separation of various N15-rGH peptides from the endogenous peptides originating from cPBMCs. Similarly, the endogenous peptides originating from rat SC tissue and rat splenocytes were also reasonably separated from N15-rGH peptides (data not shown). However, for some peptides, interference from the endogenous peptides was a major challenge while identifying the N15-rGH peptides using the fragmentation pattern. It should be noted, that when N15-rGH peptide peaks had other co-eluting endogenous peptides, it became difficult to identify the mass fragmentation pattern due to the complexity of the spectra. Therefore, N15-rGH peptides in the SC tissue, cPBMCs and rat splenocyte samples were identified using the mass spectrometry. In addition, negative control samples containing only the biological matrices were also analyzed and the peptides associated with N15-rGH were not found in those samples. Any of the identified N15-rGH peptides were not oxidized after incubation of N15-rGH with rat SC tissue, cPBMCs or rat splenocytes.

4. Discussion

In this study, we have demonstrated that ultra-pressure chromatography-mass spectrometry can be used to identify the N15-rGH peptides using tryptic digestion after incubation with various ex vivo (SC tissue) and in vitro (rat splenocytes and cPBMCs) systems. This methodology may be applied to identify oxidation products of other TPs in the SC tissue and immune cell cultures. A priori information about oxidation of TPs in the biological matrices can indicate potential of oxidation after administration. The in vitro and ex-vivo systems used here may serve as a starting point for future studies to understand oxidation of TPs after SC administration.

The purpose of this study was to identify oxidation products of N15-rGH generated by ROS originating from SC tissue and immune cell cultures, but in the protein’s host species and a model foreign host species. This hypothesis was based on known information about SC tissue and immune cells, which are sources of ROS [3–5, 12–14, 30]. The SC tissue has abundant immune cells [3]. Therefore, in this study, we did not measure ROS in the ex-vivo or in vitro systems. We assumed that the ROS may be present in the incubations. In addition, LPS was also administered to rats via SC route to increase production of ROS [15, 16]. But, after ex vivo incubation of N15-rGH with SC tissue excised from rats, the oxidation products were not detectable. Further, oxidation products of N15-rGH were not found even after 24-h incubation with cPBMCs and rat splenocytes in the presence of LPS. This could be due to low yields of oxidation or due to loss of oxidized protein via protein turnover or during sample processing steps.

In order to increase the concentration of oxidation products of the protein, hydrogen peroxide can be used as a positive control. However, in this evaluation, hydrogen peroxide was not used. Previous study from our lab evaluated oxidation of non-labeled rGH in the presence of hydrogen peroxide [2]. However, any biological matrix was not utilized. Therefore, the oxidation of N15-rGH by hydrogen peroxide remains to be studied as a positive control in the presence of biological matrix like SC tissue or immune cells.

There are few limitations of this study. First, we could not identify any oxidation products of N15-rGH. Second, this study was conducted with only one model protein and it may be useful to evaluate oxidation of other TPs like monoclonal antibodies. Third, we haven’t extensively explored in vitro systems of other immune cells (for example, macrophages, neutrophils, lymph node cells etc.). Fourth, as highlighted previously, some of the peptides were missing from the tryptic digestion of the naive N15-rGH.

Although any oxidation products of N15-rGH were not found, this is a first report describing ultra-pressure chromatography-mass spectrometry analysis of tryptic peptides of N15-rGH. This study also highlights the need of detailed evaluation of any potential oxidation of TPs after SC administration in the SC tissue and lymphatic system. In addition, the methodology reported here may be useful for identification of oxidation products of other TPs after incubation with in vitro systems or in biological matrices.

5. Conclusion

In conclusion, ultra-pressure chromatography-mass spectrometry methodology for identification of N15-rGH protein after incubation with SC tissue and immune cells was developed. The endogenous peptides originating from SC tissue and immune cells interfered with the peptides associated with N15-rGH. Although, ultra-pressure chromatography system helped to reasonably separate the peptides. The identification of peptides was possible using high-resolution mass spectrometry detector. This study provides a methodology for separation and identification of TPs after ex vivo and in vitro incubation and tryptic digestion and highlights need of further research in this field.

Supplementary Material

1
2

Highlights.

  • Rat growth hormone was identified in the biological matrices (rat subcutaneous tissue, rat splenocytes and canine peripheral blood mononuclear cells) using ultra-pressure chromatography-mass spectrometry

  • Oxidation of rat growth hormone by reactive oxygen species generated by immune cells was hypothesized, but not observed

  • This publication provides a methodology for separation and identification of therapeutic proteins after ex vivo and in vitro incubation

Acknowledgement

We thank Sharadvi Thati and Matthew Christopher for help with isolation and culture of rat splenocytes, and the Department of Pharmaceutical Chemistry for support of the mass spectrometry instrumentation.

Funding

NV and MLF were supported by a grant from the NIH (R01CA173292). NV was partially supported by the Higuchi Fellowship. We are also grateful to the J.R. and Inez Jay funds, awarded to MLF by the Higuchi Biosciences Center at The University of Kansas, for providing partial funding for this research.

Footnotes

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Supplementary Materials

1
NIHMS1750905-supplement-1.docx (3.2MB, docx)
2
NIHMS1750905-supplement-2.docx (3MB, docx)

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