Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical Formulations

Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical... Pharm Res (2018) 35: 143 https://doi.org/10.1007/s11095-018-2426-1 RESEARCH PAPER Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical Formulations 1 1 1 1 Joanna Zielińska & Jacek Stadnik & Anna Bierczyńska-Krzysik & Dorota Stadnik Received: 5 October 2017 /Accepted: 6 May 2018 /Published online: 16 May 2018 # The Author(s) 2018 . . . KEY WORDS insulin insulin analogs insulin impurities ABSTRACT Purpose Isolation and identification of unknown impurities peptide mass fingerprinting truncation of recombinant insulin lispro (produced at IBA) formed dur- ing accelerated stability testing of pharmaceutical solutions. For comparative purposes also commercially available formu- ABBREVIATIONS lations of recombinant human insulin (Humulin S®; Lilly), DTT Dithiothreitol recombinant insulin lispro (Humalog®; Lilly), recombinant EP European Pharmacopoeia insulin aspart (NovoRapid® Penfill®; Novo Nordisk), recom- HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic binant insulin detemir (Levemir®; Novo Nordisk) and recom- acid binant insulin glargine (Lantus®; Sanofi-Aventis) were IAA Iodoacetamide analyzed. IBA Institute of Biotechnology and Antibiotics, Warsaw, Methods The impurities of insulin analogs were isolated by Poland RP-HPLC and identified with peptide mass fingerprinting TFA Trifluoroacetic acid using MALDI-TOF/TOF mass spectrometry. Results The identified derivatives were N-terminally truncat- ed insulin analog impurities of decreased molecular mass of INTRODUCTION 119, 147 and 377 Da related to the original protein. The modifications resulting in a mass decrease were detected at Recombinant human insulin and its analogs are commonly the N-terminus of B chains of insulin lispro, insulin aspart, used to treat diabetes mellitus. The binding of the protein’s human insulin, insulin glargine, insulin detemir in all tested monomeric form to the insulin receptor (IR) enables regula- formulations. To our knowledge it is the first time that these tion of the blood glucose level and influences the lipid and impurities are reported. protein metabolism [1]. Nowadays, these therapeutics are Conclusions The following derivatives formed by truncation produced by recombinant DNA technology [2] and are com- of the B chain in insulin analogs were identified in pharma- mercially available as various formulations, both soluble solu- B1 B2 ceutical formulations: desPhe -N-formyl-Val derivative, tions and suspensions with protamine [3]. A detailed descrip- B1 B4 desPhe derivative, pyroGlu derivative. tion of these formulation can be found elsewhere [4–6]. In distinction to small molecule drugs, biopharmaceuticals rep- resent large, heterogeneous and complex class of medicines [7]. Their physical and chemical stability determining the Electronic supplementary material The online version of this article drug’s efficacy and safety remains a great challenge in protein (https://doi.org/10.1007/s11095-018-2426-1) contains supplementary development. In order to obtain the drug approval by regu- material, which is available to authorized users. latory authorities, recombinant therapeutics are strictly mon- itored to detect, characterize and finally eliminate or consid- * Dorota Stadnik erably limit undesirable by-products. These include deriva- stadnikd@iba.waw.pl tives formed during expression, purification and long-term storage of the biopharmaceuticals which are further evaluated Institute of Biotechnology and Antibiotics (IBA), Starościńska 5, 02-516 Warsaw, Poland in terms of toxicity and biological activity [8, 9]. Moreover, 143 Page 2 of 8 Pharm Res (2018) 35: 143 despite high structural similarity to a parent protein, chemical insulin lispro (Insulin KP drug product) was from IBA modifications induced during oxidation and the perturbation (Warsaw, Poland), recombinant insulin aspart (NovoRapid® of the secondary structure often result in enhanced immuno- Penfill®) and recombinant insulin detemir (Levemir®) were genicity of the second generation products [10, 11]. A variety from Novo Nordisk (Bagsværd, Denmark), recombinant insu- of other reported modifications includes deamidation, lin glargine (Lantus®) was from Sanofi-Aventis (Frankfurt, transamidation, racemization, oxidation, glycation, cross- Germany). links formation and disulphide scrambling [12]. The rate of derivatives formation strongly depends on the pH, tempera- Isolation of the Truncated Derivatives ture and ionic strength of the aqueous medium [13]. Also the ratio of individual components may vary depending on con- Prior to isolation all pharmaceutical formulations were incu- ditions. Most often, deamidation of insulin at residues Asn bated one month in the stability chambers at +37°C/65% A21, Asn B3, and Gln B4 is described [14–16]. The reaction RH in the original cartridges. All the above-mentioned prod- proceeds in aqueous solution under both acidic and neutral ucts differed in aging time when incubation was started. The conditions. As a result of storage in acidic pH, insulin prepa- truncated derivatives were isolated from the pharmaceutical rations deamidate primarily in AsnA21 forming AspA21 [17]. formulations by repetitive reversed phase chromatography In neutral conditions deamidation at AsnB3 occurs. It leads to (RP-HPLC) using 2695 Alliance system (Waters, Milford, the formation of AspB3 and isoAspB3 products [14]. By race- USA) equipped with 2489 UV/VIS detector (214 nm). Data mization, D-aspartyl derivatives may be produced [18–20]. acquisition and processing were conducted using Empower Moreover, Brange characterized a hydrolysis product software. 0.1 ml of the pharmaceutical formulations was resulting from the cleavage of the peptide bond between injected on two Supelcosil LC-18-dB 150 mm × 4.6 mm, 2+ ThrA8 and SerA9, occurring in Zn -rich solutions, contain- 3 μm columns connected in series. The separation was carried ing rhombohedral crystals [21]. Furthermore, the author re- out at 40°C with gradient elution at 1 mL/min, run time ports formation of covalent insulin dimers during storage of 60 min: (0–35 min) isocratic elution at A/B = 46/54; (35– pharmaceutical preparations. In solutions containing prot- 55 min) linear change to A/B = 10/90; (55–60 min) isocratic amine analogous, insulin-protamine products are formed elution at A/B = 10/90. Eluent A was 14 mM sodium per- [22]. The above processes, as well as formation of high mo- chlorate, 3.7 mM triethylamine, 4.7 mM phosphate buffer lecular weight transformation products are regarded relatively and 5.5% (vol/vol) acetonitrile, pH 2.3. Eluent B was slow in comparison to deamidation [22, 23]. Other insulin 6.5 mM sodium perchlorate, 1.7 mM triethylamine, 2.2 mM B28 derivatives determined in insulin aspart include isoAsp phosphate buffer and 50.3% (vol/vol) acetonitrile, pH 2.3. B1 B2 and desPhe -N-oxalyl-Val [16]. The latter one, resulting The peaks of the derivatives with relative retention times in 75 Da deficit, was also identified in human insulin at neutral (RRT) of 0.59; 0.78; 0.81 were collected with Fraction conditions [24]. Here we present identification of novel Collector III (Waters Milford, USA). This procedure was re- B1 B1 B2 B4 desPhe -, desPhe -N-formyl-Val - and pyroGlu insulin peated several times to obtain sufficient amount of the deriv- derivatives formed spontaneously in pharmaceutical solutions. atives from each pharmaceutical formulation. All fractions of the derivatives were pooled and evaporated to dryness using Concentrator Plus vacuum centrifuge (Eppendorf, Hamburg, MATERIALS AND METHODS Germany) and kept at −20°C until use. Chemicals Isolation of the B Chain All chemicals were of analytical reagent grade. Hydrochloric acid 35–38%, acetonitrile, sodium hydroxide were purchased The derivatives were dissolved in 0.5 ml of 0.1 M ammonium from Avantor (Center Valley, PA, USA). Sodium perchlorate, bicarbonate solution pH 8.0 and mixed with 10 μlof 0.05 M phosphoric acid 85%, trifluoroacetic acid (TFA), ammonium DTT. The mixture was incubated for 40 min at 50°C. Then carbonate were purchased from Merck (Darmstadt, 20 μl of 0.1 M IAA was added and the mixture was incubated Germany). Triethylamine, dithiothreitol (DTT), for 1 h at 25°C in darkness. Then the HPLC system (Waters iodoacetamide (IAA), formic acid, HEPES were purchased Alliance 2695, Milford, USA) equipped with a Zorbax SB- from Sigma-Aldrich (Munich, Germany). Endoproteinase C18 1.8, 50 mm × 4.6 mm column (Agilent, Santa Clara, CA, Glu-C Protease S. aureus V8 and pepsin were purchased from USA), was employed to separate and isolate the B chain. The MP Biomedicals (Santa Ana, California, USA). separation was carried out at 40°C with a linear gradient Pharmaceutical formulations: recombinant human insulin elution from 10 to 50% eluent B in 20 min at a flow rate (Humulin S®) and recombinant insulin lispro (Humalog®) 1 ml/min. Eluent A was 0.1% TFA and eluent B was 0.1% TFA with 90% ACN (both vol/vol). The peak of the B chain were from Eli Lilly (Indianapolis, IN, USA), recombinant Pharm Res (2018) 35: 143 Page 3 of 8 143 Fig. 1 Chromatogram of Insulin lispro drug product (IBA) during stability studies: after 3 years at +5°C (black line) and after 3 years at +5°C + two months at +37°C (green line). Two main peaks in this chromatogram are m-cresol (antimicrobial preservative) and insulin lispro (API). Small peaks arising from the baseline (seen in enlargement in the bottom panel) are Brelated proteins^. was collected with Fraction Collector III (Waters, Milford, USA) and eluent was evaporated as mentioned above. Enzymatic Digestion of the B Chain of Derivatives The B chains of the derivatives were dissolved in 0.1 ml of 0.1 M HEPES buffer pH 7.5 and mixed with endoproteinase Glu-C at a concentration 10 μg/ml with a mass ratio of enzyme/polypeptide chain = 1/50. The mixture was incubat- ed for 1 h at 37°C. Digestion was stopped by acidifying to pH 2 with 10% formic acid. The mixture was incubated for 15 min at 25°C. The samples were kept at −20°C until use. MALDI-TOF/TOF Mass spectra were acquired in a positive-ion reflector mode with the use of a 4800 Plus MALDI-TOF/TOF Analyzer (Applied Biosystems, Framingham, USA). Alpha-cyano-4- hydroxycinnamic acid (CHCA)fromSigma-Aldrich (Munich, Germany), dissolved in 50:50 water/acetonitrile (J.T. Baker, Deventer, The Netherlands) with 0.1% TFA – final concentration (Sigma-Aldrich, Munich, Germany), was exploited as a MALDI matrix. External calibration was achieved with a 4700 proteomics analyzer calibration mixture provided by Applied Biosystems. Samples were spotted onto a 384 Opti-TOF MALDI plate and analyzed. Data Explorer Software, Version 4.9 was applied to process acquired spectra. Mascot Distiller Software (version 2.5.1.0, Matrix Science) was employed to predict fragment ions from given peptide Fig. 2 Mass spectra of insulin lispro and its derivatives 1, 2, 3 (from top to bottom). sequences and overlay them on the acquired MS/MS spectra. 143 Page 4 of 8 Pharm Res (2018) 35: 143 TOF/TOF mass spectrometry. The recorded spectra (Fig. 2) revealed the derivatives of decreased molecular mass of 119, 147 and 377 Da related to the insulin lispro. The formation of protein impurities with mass reduced by 75 Da was observed in aspart, human, beef and pork insulin formulations [11]. B1 B2 These were desPhe -oxalyl-Val derivatives with the modi- fied Phe residue at the N-terminal of the B chain. Based on this research we predicted that isolated derivatives 1, 2 and 3 were the products of the further truncation of amino acids from the N-terminal of the B chain. Indeed, the first experi- ment involving reduction and alkylation of isolated fractions and MALDI-TOF MS analysis has shown that the observed mass decrease is due to the changes at the B chain amino acid sequence. The molecular mass of the B chain of derivative 1, 2, 3 was smaller by respectively 119, 147 and 377 Da than the molecular mass of the B chain of insulin lispro as shown in Fig. 3. To determine whether the modifications take place at the N-terminus of B chains, the B chains of all derivatives under investigation and insulin lispro were subjected to digestion with protease V8 followed by MALDI-TOF/TOF MS anal- ysis. The complete digestion of the B chain of insulin lispro with protease V8 results in three fragments: BI (B1-B13) BII (B14-B21), BIII (B22-B30) (Fig. 4). The BI peptide of insulin lispro has a monoisotopic mass of 1539.7 Da. In the derivatives’ digests, ions at m/z 1420.7 (≈1539.7–119), 1392.7 (≈1539.7–147), 1162.5 (≈1539.7– Fig. 3 Mass spectra of the B chain of insulin lispro and its derivatives 1, 2, 3 360-17) were detected and assigned to the truncated BI pep- (from top to bottom). tides of the investigated derivatives. For further structural elu- cidation, these ions were subjected to MS/MS sequencing. RESULTS AND DISCUSSION Figure 5 shows the fragmentation spectrum of m/z 1420.7, which was assigned to the BI peptide of derivative 1. All y-type ions seen in the spectrum were in agreement with theoretical, The studied insulin lispro formulations were manufactured at Institute of Biotechnology and Antibiotics with the implemen- unmodified sequence VNQHLCGSHLVE, whereas b-type ions were shifted by 119 units in comparison to the theoretical tation of recombinant DNA technology. An integral part of formulation development was stability testing which provides values for the BI peptide in insulin lispro. Based on these data, data to estimate the drug’s shelf-life and storage conditions. it was concluded that only the N-terminal amino acid residue Long-term and accelerated stability studies were performed in the BI peptide could be modified resulting in a mass de- for the insulin lispro drug product. The degradation of insulin crease by 119 Da giving the sequence as follows: lispro was monitored by RP-HPLC method and exemplary F VNQHLCGSHLVE. Upon closer inspection of the mod chromatograms obtained as a result of the analysis performed MS/MS spectrum it can be noticed that precursor ion peak are shown in Fig. 1. at m/z 1420.7 is accompanied by the cognate peak at m/z Three derivatives eluting before insulin lispro, labeled as 1, 1375.5 with a loss of 44 Da corresponding to the elimination of the formylamido group (–NHCHO) from the N-terminal of 2, 3 in Fig. 1, were isolated and characterized with MALDI- Fig. 4 Peptide fragments of the B chain of insulin lispro after digestion with protease V8. Pharm Res (2018) 35: 143 Page 5 of 8 143 Fig. 5 MS/MS spectrum of peptide B1 B1-B13 from desPhe -N-formyl- B2 Val derivative. the BI peptide. Taking into account the data presented and sequence FVNQHLCGSHLVE, the next amino acid is glu- B1 B2 analogy to previously identified desPhe -N-oxalyl-Val in- tamine (Q) which can readily cyclize to form pyroglutamate. B1 sulin, derivative 1 was recognized as desPhe -N-formyl- The process is accompanied by the loss of NH and a mass B2 Val insulin lispro. This derivative does not have the N- decrease of 17 Da [25]. In the MS/MS spectrum of BI frag- terminal NH - group at the B chain which was also confirmed ment of derivative 3 (Fig. 7), b ions corresponding to the se- by Edman degradation (see Fig. S-1 in supplementary quence with the 17 Da loss from QHLCGSHLVE were de- materials). tected beginning at b2, whereas y-series of ions were found to In case of derivative 2, the observed mass (m/z 1392.7) of be unchanged. Therefore derivative 3 was identified as fragment BI differs by 147 Da from the mass of BI of insulin pyroGluB4 insulin lispro, where the −377 Da modification lispro (m/z 1539.7) which coincides with the absence of the N- was assigned to the N-terminus of the B chain as a loss of terminal phenylalanine residue. The MS/MS spectrum of the FVN residues (−360 Da) and ammonia (−17 Da). The struc- B4 ion at m/z 1392.7 (Fig. 6) corresponds to the truncated B1 tures of pyroGlu derivative and the remaining identified peptide sequence VNQHLCGSHLVE. Based on these data derivatives are presented in Fig. 8. derivative 2 was identified as desPheB1 insulin lispro. All identified derivatives are products of truncation of N- Referring to the above result, we assumed that derivative 3 terminal residues from the B chain. What is interesting, the was also a product of truncation of N-terminal residues from truncated derivatives were detected also in the formulations of the B chain. The mass difference of 377 Da was indicative of other studied analogs (see Fig. S-2 – Fig. S-7 in supplementary the loss of three amino acid residues FVN (≈360 Da) and a materials) irrespective of the type of insulin (human insulin, moiety of 17 Da. Once the tripeptide FVN is absent from the insulin lispro, insulin aspart, insulin glargine, insulin detemir) Fig. 6 MS/MS spectrum of peptide B2-B13 from desPheB1 derivative. 143 Page 6 of 8 Pharm Res (2018) 35: 143 Fig. 7 MS/MS spectrum of peptide B4-B13 from pyroGluB4 derivative. used as the active pharmaceutical ingredient and pH of the formulation (e.g. all tested formulations have a pH 7 whereas glargine (Lantus®) has a pH of ~4). The truncation process is relatively slow in insulin pharma- ceutical solutions. Elevated levels of truncated derivatives were detected in formulations subjected to incubation at +37°C. The content of truncated insulin lispro derivatives in the insu- lin pharmaceutical formulations does not exceed EP specifi- cation limits (0.50%; any other impurity as specified in European Pharmacopoeia, monograph 01/2008:2085) dur- ing the long term stability studies at +5°C. The mechanism of truncation of proteins in insulin formulations is not fully known, however a presence of reducing agents and metal ions may play a role. The autocatalytic cleavage of peptide bond A8-A9 was observed in crystalline insulin suspensions contain- ing surplus zinc ions in addition to of those structurally bound to insulin [21]. R. Torosantucci et al. reported formation of insulin fragments during oxidation of insulin in the oxidative Cu(II)/ascorbate system [26]. Copper (II), as redox active metal ions, were also reported to be responsible for non- enzymatic fragmentation of an IgG1 monoclonal antibody [27]. Although excipients used in all tested formulations are not reducing substances themselves they can be contaminated with trace amounts of such compounds. The formation of B1 B2 desPhe -N-formyl-Val derivative can proceed by similar B1 B2 pathway as was proposed for desPhe -N-oxalyl-Val deriv- ative [16]. The transformation involves a Maillard reaction between insulin analogs and the reducing substances and sub- B1 sequent hydrolytic degradation. Both derivatives desPhe B4 and pyroGlu are products of non-enzymatic hydrolysis of insulin lispro. The latter also requires a cyclization reaction. Non-enzymatic hydrolysis of proteins and conversion of N- terminal Glu to pGlu was observed in a presence of reducing agents and metal ions [28, 29]. Therefore, we anticipate that, Fig. 8 Scheme of the N-terminal residues from B chain of (a) insulin lispro B1 these factors are the most suspicious in formation of identified (the first five residues of the B chain are shown for clarity), (b)desPhe -N- B2 B1 B4 formyl-Val derivative, (c) desPhe derivative, (d) pyroGlu derivative. truncated derivatives. Pharm Res (2018) 35: 143 Page 7 of 8 143 properties and resulting clinical outcomes. Diabetes Obes Metab. CONCLUSIONS 2017;19(1):3–12. 7. Sandra K, Vandenheede I, Sandra P. Modern chromatographic In 2002 M. U. Jars et al. published a paper on deriva- and mass spectrometric techniques for protein biopharmaceutical tives of insulin aspart [11]. One of the described deriv- characterization. J Chromatogr A. 2014;81-103(Journal Article): B1 B2 atives was desPhe -N-oxalyl-Val insulin with truncat- 8. Srebalus Barnes CA, Lim A. Applications of mass spectrometry for ed N-terminus of B chain. In our study, we present the structural characterization of recombinant protein pharmaceu- identification of consecutive derivatives of decreasing ticals. Mass Spectrom Rev. 2007;26(3):370–388. molecular mass which are formed by further truncation 9. Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M. Synthetic B1 B2 therapeutic peptides: science and market. Drug Discov Today. of thischainininsulin lispro:desPhe -N-formyl-Val 2010;15(1–2):40–56. B1 B4 derivative, desPhe derivative, pyroGlu derivative. 10. 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Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical Formulations

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Abstract

Pharm Res (2018) 35: 143 https://doi.org/10.1007/s11095-018-2426-1 RESEARCH PAPER Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical Formulations 1 1 1 1 Joanna Zielińska & Jacek Stadnik & Anna Bierczyńska-Krzysik & Dorota Stadnik Received: 5 October 2017 /Accepted: 6 May 2018 /Published online: 16 May 2018 # The Author(s) 2018 . . . KEY WORDS insulin insulin analogs insulin impurities ABSTRACT Purpose Isolation and identification of unknown impurities peptide mass fingerprinting truncation of recombinant insulin lispro (produced at IBA) formed dur- ing accelerated stability testing of pharmaceutical solutions. For comparative purposes also commercially available formu- ABBREVIATIONS lations of recombinant human insulin (Humulin S®; Lilly), DTT Dithiothreitol recombinant insulin lispro (Humalog®; Lilly), recombinant EP European Pharmacopoeia insulin aspart (NovoRapid® Penfill®; Novo Nordisk), recom- HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic binant insulin detemir (Levemir®; Novo Nordisk) and recom- acid binant insulin glargine (Lantus®; Sanofi-Aventis) were IAA Iodoacetamide analyzed. IBA Institute of Biotechnology and Antibiotics, Warsaw, Methods The impurities of insulin analogs were isolated by Poland RP-HPLC and identified with peptide mass fingerprinting TFA Trifluoroacetic acid using MALDI-TOF/TOF mass spectrometry. Results The identified derivatives were N-terminally truncat- ed insulin analog impurities of decreased molecular mass of INTRODUCTION 119, 147 and 377 Da related to the original protein. The modifications resulting in a mass decrease were detected at Recombinant human insulin and its analogs are commonly the N-terminus of B chains of insulin lispro, insulin aspart, used to treat diabetes mellitus. The binding of the protein’s human insulin, insulin glargine, insulin detemir in all tested monomeric form to the insulin receptor (IR) enables regula- formulations. To our knowledge it is the first time that these tion of the blood glucose level and influences the lipid and impurities are reported. protein metabolism [1]. Nowadays, these therapeutics are Conclusions The following derivatives formed by truncation produced by recombinant DNA technology [2] and are com- of the B chain in insulin analogs were identified in pharma- mercially available as various formulations, both soluble solu- B1 B2 ceutical formulations: desPhe -N-formyl-Val derivative, tions and suspensions with protamine [3]. A detailed descrip- B1 B4 desPhe derivative, pyroGlu derivative. tion of these formulation can be found elsewhere [4–6]. In distinction to small molecule drugs, biopharmaceuticals rep- resent large, heterogeneous and complex class of medicines [7]. Their physical and chemical stability determining the Electronic supplementary material The online version of this article drug’s efficacy and safety remains a great challenge in protein (https://doi.org/10.1007/s11095-018-2426-1) contains supplementary development. In order to obtain the drug approval by regu- material, which is available to authorized users. latory authorities, recombinant therapeutics are strictly mon- itored to detect, characterize and finally eliminate or consid- * Dorota Stadnik erably limit undesirable by-products. These include deriva- stadnikd@iba.waw.pl tives formed during expression, purification and long-term storage of the biopharmaceuticals which are further evaluated Institute of Biotechnology and Antibiotics (IBA), Starościńska 5, 02-516 Warsaw, Poland in terms of toxicity and biological activity [8, 9]. Moreover, 143 Page 2 of 8 Pharm Res (2018) 35: 143 despite high structural similarity to a parent protein, chemical insulin lispro (Insulin KP drug product) was from IBA modifications induced during oxidation and the perturbation (Warsaw, Poland), recombinant insulin aspart (NovoRapid® of the secondary structure often result in enhanced immuno- Penfill®) and recombinant insulin detemir (Levemir®) were genicity of the second generation products [10, 11]. A variety from Novo Nordisk (Bagsværd, Denmark), recombinant insu- of other reported modifications includes deamidation, lin glargine (Lantus®) was from Sanofi-Aventis (Frankfurt, transamidation, racemization, oxidation, glycation, cross- Germany). links formation and disulphide scrambling [12]. The rate of derivatives formation strongly depends on the pH, tempera- Isolation of the Truncated Derivatives ture and ionic strength of the aqueous medium [13]. Also the ratio of individual components may vary depending on con- Prior to isolation all pharmaceutical formulations were incu- ditions. Most often, deamidation of insulin at residues Asn bated one month in the stability chambers at +37°C/65% A21, Asn B3, and Gln B4 is described [14–16]. The reaction RH in the original cartridges. All the above-mentioned prod- proceeds in aqueous solution under both acidic and neutral ucts differed in aging time when incubation was started. The conditions. As a result of storage in acidic pH, insulin prepa- truncated derivatives were isolated from the pharmaceutical rations deamidate primarily in AsnA21 forming AspA21 [17]. formulations by repetitive reversed phase chromatography In neutral conditions deamidation at AsnB3 occurs. It leads to (RP-HPLC) using 2695 Alliance system (Waters, Milford, the formation of AspB3 and isoAspB3 products [14]. By race- USA) equipped with 2489 UV/VIS detector (214 nm). Data mization, D-aspartyl derivatives may be produced [18–20]. acquisition and processing were conducted using Empower Moreover, Brange characterized a hydrolysis product software. 0.1 ml of the pharmaceutical formulations was resulting from the cleavage of the peptide bond between injected on two Supelcosil LC-18-dB 150 mm × 4.6 mm, 2+ ThrA8 and SerA9, occurring in Zn -rich solutions, contain- 3 μm columns connected in series. The separation was carried ing rhombohedral crystals [21]. Furthermore, the author re- out at 40°C with gradient elution at 1 mL/min, run time ports formation of covalent insulin dimers during storage of 60 min: (0–35 min) isocratic elution at A/B = 46/54; (35– pharmaceutical preparations. In solutions containing prot- 55 min) linear change to A/B = 10/90; (55–60 min) isocratic amine analogous, insulin-protamine products are formed elution at A/B = 10/90. Eluent A was 14 mM sodium per- [22]. The above processes, as well as formation of high mo- chlorate, 3.7 mM triethylamine, 4.7 mM phosphate buffer lecular weight transformation products are regarded relatively and 5.5% (vol/vol) acetonitrile, pH 2.3. Eluent B was slow in comparison to deamidation [22, 23]. Other insulin 6.5 mM sodium perchlorate, 1.7 mM triethylamine, 2.2 mM B28 derivatives determined in insulin aspart include isoAsp phosphate buffer and 50.3% (vol/vol) acetonitrile, pH 2.3. B1 B2 and desPhe -N-oxalyl-Val [16]. The latter one, resulting The peaks of the derivatives with relative retention times in 75 Da deficit, was also identified in human insulin at neutral (RRT) of 0.59; 0.78; 0.81 were collected with Fraction conditions [24]. Here we present identification of novel Collector III (Waters Milford, USA). This procedure was re- B1 B1 B2 B4 desPhe -, desPhe -N-formyl-Val - and pyroGlu insulin peated several times to obtain sufficient amount of the deriv- derivatives formed spontaneously in pharmaceutical solutions. atives from each pharmaceutical formulation. All fractions of the derivatives were pooled and evaporated to dryness using Concentrator Plus vacuum centrifuge (Eppendorf, Hamburg, MATERIALS AND METHODS Germany) and kept at −20°C until use. Chemicals Isolation of the B Chain All chemicals were of analytical reagent grade. Hydrochloric acid 35–38%, acetonitrile, sodium hydroxide were purchased The derivatives were dissolved in 0.5 ml of 0.1 M ammonium from Avantor (Center Valley, PA, USA). Sodium perchlorate, bicarbonate solution pH 8.0 and mixed with 10 μlof 0.05 M phosphoric acid 85%, trifluoroacetic acid (TFA), ammonium DTT. The mixture was incubated for 40 min at 50°C. Then carbonate were purchased from Merck (Darmstadt, 20 μl of 0.1 M IAA was added and the mixture was incubated Germany). Triethylamine, dithiothreitol (DTT), for 1 h at 25°C in darkness. Then the HPLC system (Waters iodoacetamide (IAA), formic acid, HEPES were purchased Alliance 2695, Milford, USA) equipped with a Zorbax SB- from Sigma-Aldrich (Munich, Germany). Endoproteinase C18 1.8, 50 mm × 4.6 mm column (Agilent, Santa Clara, CA, Glu-C Protease S. aureus V8 and pepsin were purchased from USA), was employed to separate and isolate the B chain. The MP Biomedicals (Santa Ana, California, USA). separation was carried out at 40°C with a linear gradient Pharmaceutical formulations: recombinant human insulin elution from 10 to 50% eluent B in 20 min at a flow rate (Humulin S®) and recombinant insulin lispro (Humalog®) 1 ml/min. Eluent A was 0.1% TFA and eluent B was 0.1% TFA with 90% ACN (both vol/vol). The peak of the B chain were from Eli Lilly (Indianapolis, IN, USA), recombinant Pharm Res (2018) 35: 143 Page 3 of 8 143 Fig. 1 Chromatogram of Insulin lispro drug product (IBA) during stability studies: after 3 years at +5°C (black line) and after 3 years at +5°C + two months at +37°C (green line). Two main peaks in this chromatogram are m-cresol (antimicrobial preservative) and insulin lispro (API). Small peaks arising from the baseline (seen in enlargement in the bottom panel) are Brelated proteins^. was collected with Fraction Collector III (Waters, Milford, USA) and eluent was evaporated as mentioned above. Enzymatic Digestion of the B Chain of Derivatives The B chains of the derivatives were dissolved in 0.1 ml of 0.1 M HEPES buffer pH 7.5 and mixed with endoproteinase Glu-C at a concentration 10 μg/ml with a mass ratio of enzyme/polypeptide chain = 1/50. The mixture was incubat- ed for 1 h at 37°C. Digestion was stopped by acidifying to pH 2 with 10% formic acid. The mixture was incubated for 15 min at 25°C. The samples were kept at −20°C until use. MALDI-TOF/TOF Mass spectra were acquired in a positive-ion reflector mode with the use of a 4800 Plus MALDI-TOF/TOF Analyzer (Applied Biosystems, Framingham, USA). Alpha-cyano-4- hydroxycinnamic acid (CHCA)fromSigma-Aldrich (Munich, Germany), dissolved in 50:50 water/acetonitrile (J.T. Baker, Deventer, The Netherlands) with 0.1% TFA – final concentration (Sigma-Aldrich, Munich, Germany), was exploited as a MALDI matrix. External calibration was achieved with a 4700 proteomics analyzer calibration mixture provided by Applied Biosystems. Samples were spotted onto a 384 Opti-TOF MALDI plate and analyzed. Data Explorer Software, Version 4.9 was applied to process acquired spectra. Mascot Distiller Software (version 2.5.1.0, Matrix Science) was employed to predict fragment ions from given peptide Fig. 2 Mass spectra of insulin lispro and its derivatives 1, 2, 3 (from top to bottom). sequences and overlay them on the acquired MS/MS spectra. 143 Page 4 of 8 Pharm Res (2018) 35: 143 TOF/TOF mass spectrometry. The recorded spectra (Fig. 2) revealed the derivatives of decreased molecular mass of 119, 147 and 377 Da related to the insulin lispro. The formation of protein impurities with mass reduced by 75 Da was observed in aspart, human, beef and pork insulin formulations [11]. B1 B2 These were desPhe -oxalyl-Val derivatives with the modi- fied Phe residue at the N-terminal of the B chain. Based on this research we predicted that isolated derivatives 1, 2 and 3 were the products of the further truncation of amino acids from the N-terminal of the B chain. Indeed, the first experi- ment involving reduction and alkylation of isolated fractions and MALDI-TOF MS analysis has shown that the observed mass decrease is due to the changes at the B chain amino acid sequence. The molecular mass of the B chain of derivative 1, 2, 3 was smaller by respectively 119, 147 and 377 Da than the molecular mass of the B chain of insulin lispro as shown in Fig. 3. To determine whether the modifications take place at the N-terminus of B chains, the B chains of all derivatives under investigation and insulin lispro were subjected to digestion with protease V8 followed by MALDI-TOF/TOF MS anal- ysis. The complete digestion of the B chain of insulin lispro with protease V8 results in three fragments: BI (B1-B13) BII (B14-B21), BIII (B22-B30) (Fig. 4). The BI peptide of insulin lispro has a monoisotopic mass of 1539.7 Da. In the derivatives’ digests, ions at m/z 1420.7 (≈1539.7–119), 1392.7 (≈1539.7–147), 1162.5 (≈1539.7– Fig. 3 Mass spectra of the B chain of insulin lispro and its derivatives 1, 2, 3 360-17) were detected and assigned to the truncated BI pep- (from top to bottom). tides of the investigated derivatives. For further structural elu- cidation, these ions were subjected to MS/MS sequencing. RESULTS AND DISCUSSION Figure 5 shows the fragmentation spectrum of m/z 1420.7, which was assigned to the BI peptide of derivative 1. All y-type ions seen in the spectrum were in agreement with theoretical, The studied insulin lispro formulations were manufactured at Institute of Biotechnology and Antibiotics with the implemen- unmodified sequence VNQHLCGSHLVE, whereas b-type ions were shifted by 119 units in comparison to the theoretical tation of recombinant DNA technology. An integral part of formulation development was stability testing which provides values for the BI peptide in insulin lispro. Based on these data, data to estimate the drug’s shelf-life and storage conditions. it was concluded that only the N-terminal amino acid residue Long-term and accelerated stability studies were performed in the BI peptide could be modified resulting in a mass de- for the insulin lispro drug product. The degradation of insulin crease by 119 Da giving the sequence as follows: lispro was monitored by RP-HPLC method and exemplary F VNQHLCGSHLVE. Upon closer inspection of the mod chromatograms obtained as a result of the analysis performed MS/MS spectrum it can be noticed that precursor ion peak are shown in Fig. 1. at m/z 1420.7 is accompanied by the cognate peak at m/z Three derivatives eluting before insulin lispro, labeled as 1, 1375.5 with a loss of 44 Da corresponding to the elimination of the formylamido group (–NHCHO) from the N-terminal of 2, 3 in Fig. 1, were isolated and characterized with MALDI- Fig. 4 Peptide fragments of the B chain of insulin lispro after digestion with protease V8. Pharm Res (2018) 35: 143 Page 5 of 8 143 Fig. 5 MS/MS spectrum of peptide B1 B1-B13 from desPhe -N-formyl- B2 Val derivative. the BI peptide. Taking into account the data presented and sequence FVNQHLCGSHLVE, the next amino acid is glu- B1 B2 analogy to previously identified desPhe -N-oxalyl-Val in- tamine (Q) which can readily cyclize to form pyroglutamate. B1 sulin, derivative 1 was recognized as desPhe -N-formyl- The process is accompanied by the loss of NH and a mass B2 Val insulin lispro. This derivative does not have the N- decrease of 17 Da [25]. In the MS/MS spectrum of BI frag- terminal NH - group at the B chain which was also confirmed ment of derivative 3 (Fig. 7), b ions corresponding to the se- by Edman degradation (see Fig. S-1 in supplementary quence with the 17 Da loss from QHLCGSHLVE were de- materials). tected beginning at b2, whereas y-series of ions were found to In case of derivative 2, the observed mass (m/z 1392.7) of be unchanged. Therefore derivative 3 was identified as fragment BI differs by 147 Da from the mass of BI of insulin pyroGluB4 insulin lispro, where the −377 Da modification lispro (m/z 1539.7) which coincides with the absence of the N- was assigned to the N-terminus of the B chain as a loss of terminal phenylalanine residue. The MS/MS spectrum of the FVN residues (−360 Da) and ammonia (−17 Da). The struc- B4 ion at m/z 1392.7 (Fig. 6) corresponds to the truncated B1 tures of pyroGlu derivative and the remaining identified peptide sequence VNQHLCGSHLVE. Based on these data derivatives are presented in Fig. 8. derivative 2 was identified as desPheB1 insulin lispro. All identified derivatives are products of truncation of N- Referring to the above result, we assumed that derivative 3 terminal residues from the B chain. What is interesting, the was also a product of truncation of N-terminal residues from truncated derivatives were detected also in the formulations of the B chain. The mass difference of 377 Da was indicative of other studied analogs (see Fig. S-2 – Fig. S-7 in supplementary the loss of three amino acid residues FVN (≈360 Da) and a materials) irrespective of the type of insulin (human insulin, moiety of 17 Da. Once the tripeptide FVN is absent from the insulin lispro, insulin aspart, insulin glargine, insulin detemir) Fig. 6 MS/MS spectrum of peptide B2-B13 from desPheB1 derivative. 143 Page 6 of 8 Pharm Res (2018) 35: 143 Fig. 7 MS/MS spectrum of peptide B4-B13 from pyroGluB4 derivative. used as the active pharmaceutical ingredient and pH of the formulation (e.g. all tested formulations have a pH 7 whereas glargine (Lantus®) has a pH of ~4). The truncation process is relatively slow in insulin pharma- ceutical solutions. Elevated levels of truncated derivatives were detected in formulations subjected to incubation at +37°C. The content of truncated insulin lispro derivatives in the insu- lin pharmaceutical formulations does not exceed EP specifi- cation limits (0.50%; any other impurity as specified in European Pharmacopoeia, monograph 01/2008:2085) dur- ing the long term stability studies at +5°C. The mechanism of truncation of proteins in insulin formulations is not fully known, however a presence of reducing agents and metal ions may play a role. The autocatalytic cleavage of peptide bond A8-A9 was observed in crystalline insulin suspensions contain- ing surplus zinc ions in addition to of those structurally bound to insulin [21]. R. Torosantucci et al. reported formation of insulin fragments during oxidation of insulin in the oxidative Cu(II)/ascorbate system [26]. Copper (II), as redox active metal ions, were also reported to be responsible for non- enzymatic fragmentation of an IgG1 monoclonal antibody [27]. Although excipients used in all tested formulations are not reducing substances themselves they can be contaminated with trace amounts of such compounds. The formation of B1 B2 desPhe -N-formyl-Val derivative can proceed by similar B1 B2 pathway as was proposed for desPhe -N-oxalyl-Val deriv- ative [16]. The transformation involves a Maillard reaction between insulin analogs and the reducing substances and sub- B1 sequent hydrolytic degradation. Both derivatives desPhe B4 and pyroGlu are products of non-enzymatic hydrolysis of insulin lispro. The latter also requires a cyclization reaction. Non-enzymatic hydrolysis of proteins and conversion of N- terminal Glu to pGlu was observed in a presence of reducing agents and metal ions [28, 29]. Therefore, we anticipate that, Fig. 8 Scheme of the N-terminal residues from B chain of (a) insulin lispro B1 these factors are the most suspicious in formation of identified (the first five residues of the B chain are shown for clarity), (b)desPhe -N- B2 B1 B4 formyl-Val derivative, (c) desPhe derivative, (d) pyroGlu derivative. truncated derivatives. Pharm Res (2018) 35: 143 Page 7 of 8 143 properties and resulting clinical outcomes. Diabetes Obes Metab. CONCLUSIONS 2017;19(1):3–12. 7. Sandra K, Vandenheede I, Sandra P. Modern chromatographic In 2002 M. U. Jars et al. published a paper on deriva- and mass spectrometric techniques for protein biopharmaceutical tives of insulin aspart [11]. One of the described deriv- characterization. J Chromatogr A. 2014;81-103(Journal Article): B1 B2 atives was desPhe -N-oxalyl-Val insulin with truncat- 8. Srebalus Barnes CA, Lim A. Applications of mass spectrometry for ed N-terminus of B chain. In our study, we present the structural characterization of recombinant protein pharmaceu- identification of consecutive derivatives of decreasing ticals. Mass Spectrom Rev. 2007;26(3):370–388. molecular mass which are formed by further truncation 9. Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M. Synthetic B1 B2 therapeutic peptides: science and market. Drug Discov Today. of thischainininsulin lispro:desPhe -N-formyl-Val 2010;15(1–2):40–56. B1 B4 derivative, desPhe derivative, pyroGlu derivative. 10. Ottesen JL, Nilsson P, Jami J, Weilguny D, Dührkop M, Bucchini These derivatives were isolated from pharmaceutical formu- D, Havelund S, Fogh JM. The potential immunogenicity of human lations of insulin lispro produced at IBA. They were detected insulin and insulin analogues evaluated in a transgenic mouse mod- el. Diabetologia. 1994;37(12):1178–85. in the formulations of other analyzed analogs irrespective of 11. Torosantucci R, Brinks V, Kijanka G, Halim LA, Sauerborn M, the type of analog that was the active pharmaceutical ingre- Schellekens H, Jiskoot W. Development of a transgenic mouse dient in the formulation. model to study the immunogenicity of recombinant human insulin. J Pharm Sci. 2014;103(5):1367–74. 12. Manning MC, Chou DK, Murphy BM, Payne RW, Katayama ACKNOWLEDGMENTS AND DISCLOSURES DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27(4):544–575. 13. Robinson AB, Rudd CJ. 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Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem. 1987;262(2):785–794. REFERENCES 21. Brange J, Langkjaer L, Havelund S, Vølund A. Chemical stability of insulin. 1. Hydrolytic degradation during storage of pharmaceu- 1. Jirácek J, Záková L, Antolíková E, Watson CJ, Turkenburg JP, tical preparations. Pharm Res. 1992;9(6):715–726. Dodson GG, Brzozowski AM. Implications for the active form of 22. Brange J, Havelund S, Hougaard P. Chemical stability of insulin. 2. human insulin based on the structural convergence of highly active Formation of higher molecular weight transformation products hormone analogues. Proc Natl Acad Sci U S A. 2010;107(5):1966– during storage of pharmaceutical preparations. Pharm Res. 1992;9(6):727–734. 2. Mayer JP, Zhang F, DiMarchi RD. Insulin structure and function. . Brange J, Hallund O, Sørensen E Chemical stability of insulin. 5. 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Pharmaceutical ResearchSpringer Journals

Published: May 16, 2018

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