Development of Mass Spectrometry Based Techniques for the

Aug 6, 2010 - Swedish Orphan Biovitrum AB, Tomtebodavägen 23 A, SE-112 76 Stockholm, Sweden, and Symphogen A/S,. Elektrovej building 375, 2800 ...
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Anal. Chem. 2010, 82, 7274–7282

Development of Mass Spectrometry Based Techniques for the Identification and Determination of Compositional Variability in Recombinant Polyclonal Antibody Products Pia Persson,† Anders Engstro¨m,† Lone K. Rasmussen,‡,§ Erland Holmberg,† and Torben P. Frandsen*,‡ Swedish Orphan Biovitrum AB, Tomtebodava¨gen 23 A, SE-112 76 Stockholm, Sweden, and Symphogen A/S, Elektrovej building 375, 2800 Lyngby, Denmark Recombinant polyclonal antibodies are a new class of protein biologics, combining a defined number of targetspecific antibodies, developed for therapeutic use across various indications. Development, manufacture, and release of recombinant polyclonal antibodies as well characterized biological products have required development of new chemistry, manufacturing, and control (CMC) technologies. Sym001 is a recombinant polyclonal antibody product containing 25 unique antibodies specific for the Rhesus D antigen. Sym001 drug substance is manufactured using a single batch technology, Sympress. Here, we describe the development of two novel mass spectrometry based methods that allows identification of individual antibodies in the Sym001 drug substance, through the determination of unique marker peptides or antibody light chains. The two methods provide an unambiguous identification of the 25 unique antibodies comprised in the Sym001 drug substance. Furthermore, the light chain liquid chromatography-mass spectrometry (LC-MS) method has been developed to allow the determination of the relative distribution of the 25 antibodies. The light chain LC-MS method has demonstrated linearity, specificity, precision, and accuracy, thus qualifying it for use in the quality control of recombinant polyclonal antibodies for human use. The development of such quantitative methods is central for the development and quality control of additional therapeutic recombinant polyclonal antibody products. Recombinant polyclonal antibodies (rpAb) constitute a new class of therapeutic antibody products for the treatment of human diseases. In contrast to monoclonal antibodies (mAb), rpAb comprise more than one antibody species. Combining a defined number of antibodies with distinct and different specificities and mechanistic properties facilitates the development of more potent and efficacious antibody products in both infectious disease and * To whom all correspondence should be addressed. Telephone: + 45 4526 5090. Fax: + 45 4526 5060. E-mail: [email protected]. † Swedish Orphan Biovitrum AB. ‡ Symphogen A/S. § Deceased.

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cancer.1-6 mAbs and related molecules have showed a significant clinical benefit for many patients, and a substantial number of these molecules are presently in clinical development. Many mAbs have been successfully developed to deliver clinical benefit as single entities;7 however, products combining defined numbers of antibodies are presently being investigated.8 The Sym001 antibody composition was designed to reflect the natural human immune response against the Rhesus D (RhD) antigen.9 This product, thus, combines 25 specific antibodies selected for binding to the RhD antigen and relevant subtypes of the antigen. The Sym001 product is developed as an alternative to plasma derived products for the treatment of idiopathic thrombocytopenic purpura.10 mAbs are released and characterized as well characterized biologics in accordance with regulatory guidance documents and regulations.11 Release and characterization techniques for mAbs are almost generic, where new projects are being adapted to established chemistry, manufacturing, and control (CMC) analytical platforms. In contrast, the characterization of rpAb products requires the development of new innovative technologies to supplement traditional CMC analytical technologies. A technique such as analytical cation exchange chromatography (CEX) has, thus, been used to generate an overall fingerprint of antibodies present in a particular batch and to compare profiles of different product batches. CEX supports process development and provides an early assessment of rpAb characteristics. However, additional techniques are needed to identify the individual antibodies in the (1) Pedersen, M. W.; Jacobsen, H. J.; Koefoed, K.; Hey, A.; Pyke, C.; Haurum, J. S.; Kragh, M. Cancer Res. 2010, 70, 588–597. (2) Tolstrup, A. B.; Frandsen, T. P.; Bregenholt, S. Expert. Opin. Biol. Ther. 2006, 6, 905–912. (3) Bregenholt, S.; Haurum, J. Expert Opin. Biol. Ther. 2004, 4, 387–396. (4) Bregenholt, S.; Jensen, A.; Lantto, J.; Hyldig, S.; Haurum, J. S. Curr. Pharm. Des. 2006, 12, 2007–2015. (5) Haurum, J.; Bregenholt, S. IDrugs 2005, 8, 404–409. (6) Haurum, J. S. Drug Discovery Today 2006, 11, 655–660. (7) Reichert, J. M.; Rosensweig, C. J.; Faden, L. B.; Dewitz, M. C. Nat. Biotechnol. 2005, 23, 1073–1078. (8) Swann, P. G.; Tolnay, M.; Muthukkumar, S.; Shapiro, M. A.; Rellahan, B. L.; Clouse, K. A. Curr. Opin. Immunol. 2008, 20, 493–499. (9) Andersen, P. S.; Haahr-Hansen, M.; Coljee, V. W.; Hinnerfeldt, F. R.; Varming, K.; Bregenholt, S.; Haurum, J. S. Mol. Immunol. 2007, 44, 412– 422. (10) Andersson, P. O.; Wadenvik, H. Expert Rev. Mol. Med. 2004, 6, 1–17. (11) Kozlowski, S.; Swann, P. Adv. Drug Delivery Rev. 2006, 58, 707–722. 10.1021/ac101175w  2010 American Chemical Society Published on Web 08/06/2010

rpAb drug substance, to verify the polyclonality, and to evaluate the compositional variability in rpAb products. The use of various mass spectrometry (MS) based techniques for the analysis of complex biological products has increased in recent years.12 MS based techniques are used extensively for the analysis of product related substances and contaminants, different types of post-translational modifications, N-linked oligosaccharides, and the verification of primary sequence and potential sequence variants across all types of therapeutic proteins.12,13 MS based methods are also more frequently being developed and used for the quality control and release of complex biologics, including mAbs,14,15 i.e., for the verification of the primary structure, the arrangement of the disulfide bridges, the structure and profile of N-linked carbohydrate, post-translational modifications, and potential other modifications.15 Here, we describe the development of MS based methods for the analysis and identification of individual antibodies in an rpAb product. We have developed methods that allow the identification of antibodies and for determination of the relative distribution of individual antibodies in an rpAb product containing 25 RhD specific antibodies. Such techniques have been used for additional characterization of manufactured rpAb and have the potential to be applied as future release methods. EXPERIMENTAL SECTION Materials. All chemicals were of analytical grade. Guanidine hydrochloride (Gnd-HCl), DL-dithiothreitol (DTT), Tris, iodoacetic acid, urea, sodium hydro carbonate, sodium phosphate (monoand dibasic), ammonium acetate, and propionic acid were purchased from Sigma Aldrich (St Louis, MO). Trifluoroacetic acid (TFA) was purchased from Thermo Scientific (Rockford, IL). High performance liquid chromatography (HPLC) grade acetonitrile and isopropanol were obtained from LabScan Analytical Sciences (Dublin, Ireland). Sequencing grade endoproteinase Asp-N was obtained from Roche (Basel, Switzerland). Cultivation and Protein Purification. Cultivation and purification of an rpAb containing 25 RhD specific antibodies were performed essentially as described.16 Sym001 was initially manufactured from a polyclonal master cell bank (pMCB)16 and subsequently from a polyclonal working cell bank (pWCB) where small changes to the manufacturing process have also been included. Individual monoclonal antibodies were cultivated and purified employing a MabSelect or a MabSelect SuRe column (GE Healthcare, Bucks, UK) and dialyzed against 5 mM sodium acetate, 150 mM NaCl, pH 5, essentially as described.16 Reduction and Carboxymethylation. Sym001 samples and individual antibodies were desalted using a PD-10 column (GE Healthcare) equilibrated in water or with dialysis (Slide-A-Lyzer dialysis cassettes, Thermo Scientific, IL) against water. The samples were dried in a vacuum centrifuge and subsequently reconstituted in 6 M Gnd-HCl, 0.2 M Tris, pH 8.4, to a final (12) Domon, B.; Aebersold, R. Science 2006, 312, 212–217. (13) Srebalus Barnes, C. A.; Lim, A. Mass Spectrom. Rev. 2007, 26, 370–388. (14) Houde, D.; Kauppinen, P.; Mhatre, R.; Lyubarskaya, Y. J. Chromatogr., A 2006, 11, 189–198. (15) Zhang, Z.; Pan, H.; Chen, X. Mass Spectrom. Rev. 2009, 28, 147–176. (16) Wiberg, F. C.; Rasmussen, S. K.; Frandsen, T. P.; Rasmussen, L. K.; Tengbjerg, K.; Coljee, V. W.; Sharon, J.; Yang, C. Y.; Bregenholt, S.; Nielsen, L. S.; Haurum, J. S.; Tolstrup, A. B. Biotechnol. Bioeng. 2006, 94, 396– 405.

concentration of approximately 10 mg/mL. Reduction was performed by addition of 0.005 µL 1 M DTT/µg protein followed by incubation at 65 °C for 30 min. The sample was subsequently alkylated by addition of 0.012 µL of 1 M iodoacetic acid/µg protein and incubated in the dark for 40 min at room temperature. The reaction was then quenched by addition of 0.003 µL of 1 M DTT/ µg protein. Separation of Heavy and Light Chain with Gel Filtration Chromatography. The heavy and light chains of reduced and alkylated samples were separated on a Superose 12 column (10 × 300 mm; GE Healthcare) in 6 M Gnd-HCl, 50 mM sodium phosphate, pH 8.4, operated at room temperature using a flow rate of 0.1 or 0.15 mL/min. Approximately 2 mg of Sym001 or 0.5-2 mg of individually produced antibodies was applied onto the column. The separated heavy and light chains were detected at 280 nm. Fractions containing light chains were pooled and dialyzed against 0.1 M ammonium acetate. Individually produced antibody light chains were dialyzed against 0.1 M ammonium acetate or 1 M urea, 50 mM sodium phosphate, pH 8.0. Fractions containing heavy and light chains to be enzymatically digested were dialyzed against 1 M urea, 50 mM sodium phosphate, pH 8.0. To prevent carbamylation, the urea was purified by adding 50 g of ion-exchange resin AG-501-X8 (Bio-Rad, Hercules, CA) to 1000 mL of a 6 M urea stock solution and stirring on a magnetic stirrer for 5 h. Proteolytic Digestion. Endoproteinase Asp-N was added to the light and heavy chain preparations in a final ratio of 1:200 (enzyme/protein) and incubated at 37 °C for 16 h. The enzymatic reaction was quenched by addition of TFA to a final concentration of 0.5% (v/v). Reversed Phase-HPLC/MS Analysis of Marker Peptides. Reversed phase (RP)-HPLC/MS of the proteolytic digestion was carried out on an Agilent 1100 HPLC system coupled to an Agilent 1100 LC/MSD mass spectrometer. The enzymatic digest (10-15 nmole) was applied onto a Zorbax 300SB-C18 column (2.1 × 150 mm, Agilent, Santa Clara, CA) in 80% mobile phase A (water, 0.1% TFA) and 20% mobile phase B (acetonitrile, 0.08% TFA) at 40 °C using a flow rate of 0.2 mL/min. The peptides were separated using a gradient of 20-55% mobile phase B in 290 min for light chain peptides and 360 min for heavy chain peptides, followed by a gradient of 50-100% B in 30 min. Peptides were detected with electrospray ionization-quadropole (ESI-q) MS. The mass spectrometer was set up to run in a positive ion mode with an m/z range of 400-2000. A mixture of 75% propionic acid and 25% isopropanol was added to the mobile phase prior to mass detection at a flow rate of 10 µL/min for fixation of TFA. The obtained MS data were analyzed using GPMAW 6.2 (Lighthouse data, Odense, Denmark) or Masslynx 3.5 (Waters, Milford, MA). N-Terminal Sequencing. Collected peptides were prepared with Prosorb sample preparation cartridges (Applied Biosystems, Carlsbad, CA) according to the manufacturer and sequenced on a Procise 494 sequenator (Applied Biosystems) which automatically perform N-terminal sequencing according to the Edman degradation. Reversed Phase-High Performance Liquid Chromatography and MS Analysis of Intact Light Chains. Reversed phasehigh performance liquid chromatography (RP-HPLC) and MS of intact light chains were performed on an Agilent 1100 HPLC Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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system connected to an Agilent MSD TOF mass spectrometer. A light chain preparation (about 50 µg) was applied onto an ACE 3 C4-300 column (2.1 × 100 mm; Advanced Chromatography Technologies, Aberdeen, UK) in 69% mobile phase A (water, 0.05% TFA) and 31% mobile phase B (acetonitrile, 0.04%TFA) at 60 °C using a flow rate of 0.4 mL/min. The gradient was 31-38% mobile phase B in 70 min, and the light chains were detected at 214 nm and using electrospray ionization-time-of-flight (ESI-TOF) MS. The mass spectrometer was set up to run in a positive ion mode with an m/z range of 300-2800. Theoretical masses of the light chains, based on amino acid sequences, and m/z values were calculated with MassLynx 3.5. Extracted ion chromatograms (XICs) and m/z spectra were obtained using Analyst QS 1.1 software (Applied Biosystems). Deconvolutions were performed with Agilent BioConfirm A.02.00 software. Safety Considerations: Acetonitrile is highly flammable and is, furthermore, harmful by contact, inhalation, and ingestion. Guanidine hydrochloride is harmful if swallowed and causes irritation to eyes and skin. Iodoacetic acid is toxic and causes irritation to the skin. NaOH is corrosive and causes severe burns. TFA is extremely corrosive and volatile. TFA is, furthermore, harmful by contact, inhalation, or ingestion. RESULTS AND DISCUSSION Development of a Marker Peptide Method. Different approaches were evaluated to identify a MS based method for identification of the individual antibodies in Sym001. The first approach relied on identification of antibody-specific peptides (marker peptides) in mixtures obtained by digestion using sequence specific proteases. Such marker peptides would originate from the variable parts located at the N-terminal parts of the light and heavy chains of the antibodies. Detailed theoretical analysis of the amino acid sequences revealed that digestion using endoproteinase AspN, which specifically cleaves at the N-terminus of aspartate (Asp), generally generated relatively large and more hydrophobic peptides from the variable regions of both the light and the heavy chain as compared to peptides from the remaining part of the sequence. Thus, most likely, this difference in physicochemical properties of the individual peptides could be used for the development of a suitable method, allowing separation of unique N-terminal marker peptides from remaining peptides. To decrease the complexity of the obtained peptide mixture, the heavy and light chains were separated by gel filtration chromatography, following reduction and alkylation of the Sym001 full length antibodies. The isolated pools of heavy and light chains were subsequently desalted and digested using endoproteinase Asp-N, separated by RP-HPLC, and analyzed using ESI-q MS. During early method development, the gel filtration fractions were buffer exchanged into 0.1 M ammonium hydro carbonate (pH 8.0). In this buffer, however, both light chain and heavy chain preparations precipitated after digestion. Addition of 1 M urea, 10% (v/v) acetonitrile, or 1 M Gnd-HCl to the buffer was tested, and with any one of these additions, the peptides remained in solution. The most efficient digestion and the highest coverage of peptides were obtained with a buffer consisting of 0.05 M sodium phosphate, 1 M urea (pH 8.0). Subsequent evaluation of the liquid chromatography-mass spectrometry (LC-MS) data revealed that some peptides originating from nonspecific cleavage at glutamate (Glu) were obtained. Of the two tested enzyme/ 7276

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substrate ratios, i.e., 1:100 and 1:200, and digestion overnight, a ratio of 1:200 was chosen to minimize nonspecific digestion. The developed method showed that masses corresponding to specific peptides originating from all 25 antibodies in Sym001 could be detected. More than one specific peptide was identified for 18 of the antibodies in Sym001. One peptide for each antibody was chosen as a marker peptide, and most of these peptides were relatively hydrophobic in character based on their retention times in an HPLC analysis (Table S-1 and Figure S-1, Supporting Information). All peptides were originating from the light chain except one peptide, corresponding to antibody RhD293, which originated from the heavy chain. The masses of three of the N-terminal marker peptides (RhD197 d1, RhD207 d1, and RhD319 d1), harboring an Nterminal glutamine (Gln), corresponded to masses where the Gln residue was cyclized to pyroglutamic acid (pyroGlu; -17 Da), a well-known post-translational modification in antibodies.17 The specificity of the identified marker peptides was tested by mass searches in all 25 antibodies with the software GPMAW with a precision of 400 ppm. The following parameters were considered: cleavage specificity on the N-termini of both Asp and Glu residues, missed cleavage sites, cyclization of the N-terminus for antibodies with an N-terminal Gln, and three possible known glycosylation patterns of the heavy chain (fucosylated complex biantennary G0 (G0F), fucosylated complex biantennary G1 (G1F), or fucosylated complex biantennary G2 (G2F)). Other possible post-translational modifications were not investigated. Experimental masses corresponding to peptides RhD159 d1, RhD191 d1, RhD196 d2, RhD301 d1, RhD305 d1, and RhD321 d1 also matched other theoretical peptides in the enzymatic digest of the 25 antibodies with these search criteria. In such cases, peptides were identified and confirmed by N-terminal sequencing except for peptide RhD196 d2. This light chain peptide had the same mass as a peptide in the heavy chain. However, since the light chain peptide was not found at this retention time in runs of separated heavy chain peptides, the identity of the peptide was assigned as RhD196 d2. It may be questioned if one peptide is sufficient to identify a protein, but as this is a recombinantly produced mixture of 25 known antibodies, the probability for misidentification is extremely low as compared to, e.g., proteomics where the protein mixture is far more complex and where more than one peptide typically is needed to identify a protein. However, misidentification due to unknown post-translational modifications cannot be excluded. Possible carbamylation of lysine (+43 Da) due to the presence of urea during sample preparation was investigated but not detected. We have also not identified mass changes due to over- or underalkylation during analysis of individually produced antibodies. We have confirmed the presence of all 25 Sym001 antibodies in four individual industrial scale batches using the marker peptide method. These data confirm previous observations on active pharmaceutical ingredient (API) consistency between batches16 and demonstrate that all antibodies are recovered from the downstream manufacturing process at a relevant scale. LC-MS of Intact Light Chains. The developed marker peptide method provided a unique identification of the 25 antibodies in Sym001; however, the obtained results were qualitative, only providing an identification of the presence of a specific antibody. (17) Jones, G. H. Biochemistry 1974, 13, 855–860.

Table 1. Mass Accuracy of Three Molecular Ions and Retention Times of Light Chains of Individually Produced Antibodies and Antibody Light Chains in a Sym001 Working Standard, Run in Parallel mass accuracy (ppm) individual antibodies antibody light chains in mass order

theoretic massa(Da)

RhD 207c RhD 207d RhD 197d RhD 203d RhD 319d RhD 160 RhD 162 RhD 192 RhD 245 RhD 321 RhD 159 RhD 293 RhD 317 RhD 305 RhD 199 RhD 202 RhD 201 RhD 324 RhD 196 RhD 241 RhD 240 RhD 189 RhD 157 RhD 306 RhD 191 RhD 301

22861.57 23043.75 23143.86 23179.57 23288.94 23334.93 23384.00 23432.00 23590.09 23640.98 23660.15 23684.29 23702.29 23705.22 23733.23 23742.27 23792.42 23813.40 23822.42 23845.48 23856.44 23889.51 23997.70 24004.78 24169.69 24280.06

Sym001

retention time (minutes)

∆Mb

ion 1

ion 2

ion 3

ion 1

ion 2

ion 3

Ind. Ab

Sym001

182 100 36 109 46 49 48 158 51 19 24 18 3 28 9 50 21 9 23 11 33 108 7 165 110

13 20 20 3 54 -5 -3 44 46 1 37 34 47 30 21 40 -7 47 21 65 17 25 16 3 6 14

15 17 15 -2 47 -4 -2 45 42 5 37 31 48 29 20 41 -4 42 24 58 21 18 25 -1 6 16

13 13 7 2 44 -7 -5 41 39 -4 37 23 45 35 24 35 -3 38 19 54 16 18 20 1 4 12

27 14 12 2 23 -5 -7 33 43 -12 36 30 39 33 12 40 -6 45 18 46 15 24 19 2 0 5

22 13 9 11 24 -4 -2 34 42 1 36 8 43 16 13 37 -4 49 16 41 13 6 17 -5 -3 -11

15 13 7 6 -10 -4 -3 31 33 36 33 19 43 9 12 36 -3 51 16 39 13 -37 16 -4 -3 10

41.3 42.6 44.4 12.8 55.2 32.5 54.8 35.7 38.0 19.5 19.4 50.8 35.8 16.1 30.0 16.1 35.3 20.0 24.3 27.8 35.1 28.8 27.6 30.8 34.3 31.1

41.3 42.6 44.1 12.7 55.3 31.2 54.8 35.3 38.1 18.7 18.8 50.8 36.0 15.4 28.0 15.5 34.5 20.0 23.3 26.5 34.4 27.8 25.4 29.6 32.2 29.3

a Calculation of theoretic mass was based on the sequences of antibody light chains. Cysteine residues were carboxymethylated. b ∆M is the mass difference between the present light chain and the light chain with the closest mass below the present light chain. c A truncated form of RhD 207 lacking the two first N-terminal amino acids (-QA). d N-Terminal glutamine was cyclized to pyroglutamic acid.

We, therefore, aimed at developing an MS based method allowing quantification of the individual antibodies in Sym001. For this purpose, the light chains of the antibodies were chosen for several reasons. First, the light chains represent the distribution of full length antibodies since the ratio of light and heavy chains was expected to be 1:1. Second, the mass resolution for smaller molecules is higher. The mass of the Sym001 light chains is approximately 24 000 Da as compared to the mass of full length antibodies of 150 000 Da. Moreover, in contrast to the heavy chains, the light chains in the Sym001 product are not glycosylated. Glycosylation leads to heterogeneity in molecular mass which increases the complexity of m/z spectra. Finally, the light chains have larger regions with more unique sequences. Analysis of the theoretical sequences of the 25 individual light chains showed that their masses were unique, potentially allowing identification based solely on mass. The theoretical masses of the 25 light chains are listed in Table 1 in mass order. Mass differences less than about 10 Da were not resolved by the mass spectrometer, and chromatographic resolution in the HPLC step was necessary to resolve such antibody light chains. Again, to decrease the complexity of the method and of the subsequent MS data, the Sym001 sample was separated into pools of heavy and light chains using gel filtration chromatography. The isolated pool of light chains was desalted, separated by RP-HPLC, and analyzed using ESI-TOF MS. Desalting prior to the LC-MS step was necessary to prevent potential guanidium adduct formation. The RP-HPLC separation of the different antibody light chains was optimized by testing different columns, column temperatures,

acetonitrile gradients, and mobile phase flow rates. A representative chromatogram where optimal resolution was obtained for all critical antibody light chains is shown in Figure 1a (total ion counts, TIC) and Figure 1b (UV), and a deconvoluted mass spectrum for a time frame including all the peaks in the TIC is shown in Figure 1g. For identification of the antibody light chains, theoretical m/z values were extracted to generate extracted ion chromatograms (XIC) of each light chain constituting Sym001. The three ions with the highest intensity in the charged envelope were compared with theoretical m/z values. XICs and m/z spectra for antibody light chains of a high concentration antibody (RhD 157) and a low concentration antibody (RhD 203) are shown in Figure 1c-f. For relative quantification, a second XIC generated from the experimentally obtained m/z value of one ion, the ion with the highest intensity, was integrated after smoothing. The relative quantification was then expressed as relative percent for each light chain. Qualification of the Light Chain LC-MS Method. The light chain LC-MS method has been further qualified for use in Sym001 manufacturing quality control. The method was qualified using a number of parameters such as specificity, linearity, precision, and accuracy. The specificity of the method was tested by analysis of the 25 individually produced antibodies in parallel with a Sym001 working standard. All antibodies contained a light chain of the expected mass (Table 1) except a number of antibodies with known post-translational modifications or truncations. The analysis of the individual antibodies, thus, showed that the main masses obtained for the light chains with an N-terminal Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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Figure 1. RP-HPLC separation of light chains in Sym001 and evaluation of antibodies RhD 157 and RhD 203. (a) total ion counts chromatogram; (b) UV (λ ) 214 nm) chromatogram; (c) extracted ion chromatogram of the RhD 157 light chain. One theoretical molecular ion, m/z 960.92 (M + 25H+), was extracted with a mass window of m/z 0.2, corresponding to a mass window of 5 Da; (d) extracted ion chromatogram of the RhD 203 light chain. One theoretical molecular ion, m/z 1054.62 (M + 22H+), was extracted with a mass window of m/z 0.2, corresponding to a mass window of 4.4 Da. The two later eluting peaks originate from RhD191 (m/z 1051.9) and RhD197 (m/z 1053.0); (e) m/z spectrum of RhD 157 light chain; (f) m/z spectrum of RhD 203 light chain; (g) deconvoluted mass spectrum of total ion count chromatogram, 13-58 min. RhD 199 was not resolved from RhD 202, and RhD 321 and RhD 324 were low concentration antibodies and were not resolved/detected in the deconvoluted mass spectrum. 7278

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Figure 2. Linearity for different injected amounts of a Sym001 working standard. Linearity for (a) a high concentration antibody (RhD 157), (b) a low concentration antibody (RhD 203), and (c) the sum of all 25 antibodies. (d) Shows the relative area% for each antibody light chain at the different levels.

Gln residue corresponded to masses of pyroGlu forms, as was also found using the marker peptide method. It was also demonstrated that about 50% of the light chains of antibody RhD 207 were truncated with two amino acids in the N-terminus. Analysis of the individually produced antibodies also showed that masses corresponding to other post-translational modifications were not found in amounts that would influence the quantification. Other potential protein modifications with small or no mass difference like deamidation of asparagine and isomerization of aspartic acid were not resolved by this method. We have studied the potential presence of such modifications using other methods and have not, e.g., detected isoaspartic acid in Sym001 using an HPLC based detection kit. The masses obtained for individual

antibody light chains corresponded very well with masses found in the Sym001 sample. The identities of the antibody light chains in Sym001 were further confirmed by their retention times which were in agreement with retention times obtained in runs of the individual light chains (Table 1). Thus, misidentification is very unlikely since both mass and retention time was used to identify the antibodies. The linearity of the integrated extracted ion peaks for the antibody light chains in the Sym001 working standard was tested by construction of plots with five levels between 50% and 150% of a standard sample load of 50 µg. Three replicate injections were performed at each level. Correlation coefficients were between 0.9807 and 0.9946, except for three low concentration antibodies Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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Table 2. Relative Quantification of Antibodies Constituting Sym001 Sym001 working standard, n ) 10

Sym001 MYS08-02, n)3

antibody light chain

relative area%

std. dev

RSD%

relative area%

std. dev

RSD%

RhD 157 RhD 159 RhD 160 RhD 162 RhD 189 RhD 191 RhD 192 RhD 196 RhD 197a RhD 199 RhD 201 RhD 202 RhD 203a RhD 207b RhD 240 RhD 241 RhD 245 RhD 293 RhD 301 RhD 305 RhD 306 RhD 317 RhD 319a RhD 321 RhD 324

15.2 4.3 21.3 1.7 0.6 7.2 1.5 3.8 3.6 1.9 4.7 9.6 0.3 5.3 1.8 3.0 1.0 0.8 1.8 3.0 4.9 1.1 1.1 0.2 0.3

0.35 0.12 0.66 0.11 0.04 0.15 0.06 0.10 0.10 0.07 0.17 0.31 0.03 0.26 0.05 0.06 0.07 0.04 0.07 0.12 0.22 0.02 0.05 0.02 0.03

2.3 2.8 3.1 6.6 6.2 2.1 3.8 2.6 2.6 3.7 3.5 3.2 7.8 4.9 2.6 1.9 6.7 4.8 4.2 4.0 4.5 1.9 4.4 9.1 10.2

11.1 3.1 20.7 0.9 3.1 9.5 1.5 2.9 3.4 0.9 4.7 19.4 0.1 4.5 0.8 1.6 1.4 0.5 4.4 2.1 2.0 0.6 0.4 0.2 0.1

0.40 0.06 0.58 0.04 0.10 0.22 0.03 0.09 0.14 0.05 0.09 0.27 0.03 0.20 0.01 0.02 0.04 0.02 0.10 0.03 0.03 0.03 0.01 0.01 0.02

3.6 1.9 2.8 4.8 3.4 2.3 2.0 3.2 4.2 5.7 2.0 1.4 19.9 4.5 0.7 1.1 2.8 4.3 2.3 1.2 1.7 4.4 3.3 2.2 13.3

a N-Terminal glutamine was cyclized to pyroglutamic acid. b The sum of two forms of RhD207 is reported. About 50% has an N-terminal glutamine cyclized to pyroglutamic acid, and about 50% lacks the two first N-terminal amino acids.

RhD 203, RhD 321, and RhD 324 which had correlation coefficients of 0.9730, 0.9583, and 0.9744, respectively. Plots for a high concentration antibody (RhD 157) and a low concentration antibody (RhD 203) are shown in Figure 2a,b. A plot with the sum of responses for all antibodies is shown in Figure 2c. It was noticed that the response decreased slightly during a sequence run. This is shown by a decrease in the responses of the 50 µg injection which were run in the beginning, the middle, and the end of the sequence while the other levels were injected repeatedly (Figure 2a-c). A possible explanation for the observed decreases in response is that the MS inlet becomes blocked due to the high sample load of 50 µg and the relatively high flow rate of 0.4 mL/minute for a 2 mm diameter column. The high flow rate was chosen to obtain a better selectivity, and the high sample load was necessary to allow detection of some low concentration antibodies. It is possible that a smaller column diameter together with a lower sample load would overcome such decreased response. In the present study, however, the decrease in response did not affect the relative quantification of antibodies at the different levels (Figure 2d). The relative standard deviation (RSD) values were below 6% for all antibody light chains except for RhD 321, which showed a higher RSD value (13.2%) likely explained by its relatively small area. The relative quantification of antibodies in 10 independent analyses of the Sym001 working standard is shown in Table 2. The intermediate precision was high with RSD values between 1.9% and 10.2% for the different light chains, where the higher values were obtained for low concentration antibodies. To show 7280

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that differences in the antibody distribution could be detected reproducibly, another batch of Sym001 (MYS08-02) was analyzed in parallel with the Sym001 working standard at three independent occasions. For MYS08-02, RSD values were below 10%, except for two antibodies, RhD 203 and RhD 324, that constituted low amounts of around 0.1% (Table 2). Equimolar amounts of different compounds do not necessarily give equal responses in MS because only a fraction of the molecules is ionized and this fraction depends partly on the molecular structure. To achieve absolute quantification, the mass spectrometer should be calibrated with known amounts of the compound to be measured, but this was not feasible for measuring the 25 light chains in Sym001. However, we believe that the specific response of the individual antibody light chains would be fairly similar since the antibodies were homologous, also in the variable regions.9 The relative ionization efficiency of the antibody light chains was investigated by comparing total ion counts (TIC) in runs of individually produced antibodies with the signals obtained at 214 nm, where the absorbance of peptide bonds are measured and which is an accurate measurement of protein concentration. Since the response of the mass detector was not reproducible, the TIC signal was normalized against the response of one of the light chains (RhD 160) in the Sym001 working standard, run in parallel at each analysis occasion. The TIC/λ214 ratio (based on normalized TIC responses) was calculated for all light chains (Table 3). The average value was 0.17 counts × 106/ mAU × min, with an RSD value of 19%. Furthermore, quantification of selected antibody light chains in the Sym001 working standard by integration of UV peaks showed that similar relative areas as with XICs were obtained (Table 4). We also showed that integration of only one molecular ion in the charge envelope was a good estimation for the total response by measuring the TIC/XIC ratio in runs of the individual antibodies with a calculated average value of 39 (RSD of 11%) (Table 3). To further investigate the accuracy of the method, spike-in experiments using individual light chains were performed. The light chain of each individual antibody was prepared, and the concentration was determined by measuring the absorbance at 280 nm. The light chains were then analyzed individually at one or two concentration levels and spiked to the Sym001 working standard at the same two levels. A run of Sym001 without spiked light chains was performed in parallel. For calculation of recovery and linear regression, the observed decrease in response during sequences described above was compensated for by the use of at least three of the light chains in the unspiked Sym001 sample as an “internal standard”. The recovery results of spike-in samples are shown in Table 3, and plots for RhD 157, RhD 203, and RhD 324 are shown in Figure 3. The recovery was between 69% and 134%. The high recovery for RhD 324 LC (134% and 128% for the two levels) were due to underestimation of added antibody light chain, since the response for the higher concentration level was lower than expected (Figure 3c). The reason for this lower response was not known. The linearity was good for all the individual light chains in spike-in samples (R2 > 0.99). The linearity for the light chains run individually was also good with R2 values >0.99 for all antibodies except RhD 317 (0.9860) and RhD 324 (0.9646).

Table 3. Accuracy in Relative Quantification of the Light Chain LC-MS Method recoveryb (%) antibody light chain

ratio norm. TIC/λ214a [(counts*106)/(mAU*min)]

ratio TIC/XICa

RhD 157 RhD 159 RhD 160 RhD 162 RhD 189 RhD 191 RhD 192 RhD 196 RhD 197c RhD 199 RhD 201 RhD 202 RhD 203c RhD 207d RhD 240 RhD 241 RhD 245 RhD 293 RhD 301 RhD 305 RhD 306 RhD 317 RhD 319c RhD 321 RhD 324 average RSD%

0.14 0.18 0.19 0.20 0.17 0.22 0.14 0.14 0.13 0.12 0.19 0.17 0.14 0.14 0.13 0.19 0.19 0.16 0.18 0.17 0.21 0.19 0.12 0.12 0.21 0.17 19

29 33 39 41 38 47 41 41 44 46 37 38 44 43 38 34 38 42 45 38 40 38 37 38 35 39 11

linearity (R2)

level 1

level 2

antibody light chain alone

antibody light chain in Sym001

88 121 98 80 108 81 120 104 69 123 114 98 77 84 104 104 122 132 94 71 85 97 98 95 134

101 112 101 80 107 74 121 101 79 112 102 87 81 86 119 106 117 121 95 78 79 88 101 104 128

0.9935 1.0000 n.d.e n.d.e 0.9952 0.9970 0.9999 0.9977 0.9996 0.9994 n.d.e 0.9971 0.9995 0.9997 1.0000 1.0000 0.9971 0.9956 n.d.e 0.9953 n.d.e 0.9860 0.9986 n.d.e 0.9646

0.9943 0.9980 0.9914 1.0000 1.0000 0.9909 1.0000 0.9998 0.9936 0.9968 0.9926 0.9943 0.9970 0.9998 0.9944 0.9999 0.9992 0.9972 1.0000 0.9974 0.9995 0.9960 0.9998 0.9965 0.9994

a The TIC/λ214 ratios and the TIC/XIC ratios were determined from runs of light chains of individually produced antibodies. b Recovery was calculated according to the following formula:

recovery (%) )

(normalized response in spiked sample - normalized response in unspiked sample) × 100 response of antibody light chain, run alone (average)

c

N-Terminal glutamine was cyclized to pyroglutamic acid. d The sum of two forms of RhD207 is reported. About 50% has an N-terminal glutamine cyclized to pyroglutamic acid, and about 50% lacks the two first N-terminal amino acids. e n.d.: Not determined.

were 5.3% and 1.8%, respectively (n ) 10), which further confirms the reliability in the LC-MS data.

Table 4. Comparison of XIC and UV Responses of Antibody Light Chains in Sym001 Working Standard XIC (n ) 10) RhD 159 RhD 196 RhD 197 RhD 245 RhD 293

UV (n ) 3)

relative area%

RSD%

relative area%

RSD%

4.3 3.8 3.7 1.0 0.8

2.8 2.6 2.6 6.7 4.8

3.6 4.4 4.8 0.9 0.7

8.1 5.7 0.0 17.6 8.7

The results for two of the antibodies (RhD 207 and RhD 240) using the light chain LC-MS method was compared with results obtained with an CEX method16 where full-length antibodies were analyzed and detected spectrophotometrically at 214 nm. This method has been used extensively during process development and to compare batches produced at an industrial scale.16 With this method, antibodies RhD 207 and RhD 240 were baseline separated from other antibodies, and the relative amount of RhD 207 and RhD 240 in the Sym001 working standard was 5.4% and 1.4%, respectively (n ) 9, RSD was 5.4% and 8.3%, respectively). These values were in agreement with the obtained results with the LC-MS method which

CONCLUSIONS We have described the successful development of an MS based method to identify and quantify all 25 antibodies in a rpAb product Sym001 based on differences in the molecular weight of individual light chains. The method identifies light chains based on retention times in the HPLC separation step and, furthermore, based on their unique masses. For relative quantification of the individual light chains, XICs were generated from the experimentally obtained m/z value of the ion with the highest intensity and the relative quantification of the 25 antibodies was then expressed as relative percent for each light chain. The reported light chain LC-MS method has provided data on the individual antibodies in various Sym001 batches. As expected, the present light chain LC-MS data have confirmed an appropriate high degree of consistency of the API between batches previously reported;16 however, the data also reveal some variation in the levels of individual antibodies in the Sym001 drug substance. The light chain LC-MS method for identification and quantification of rpAb is believed to be an important tool for Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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tions to be set for rpAb products based on the method qualification evaluation and manufacturing data. The reported method is generic to some extent. This method will, thus, most likely be useful for analysis of newly developed rpAb products, provided that such a product contains antibodies with discernible light chains. Only in the very rare instances where light chains will have the same molecular mass and are not possible to separate by HPLC will the method have some limitations. We have demonstrated that rpAb products can be manufactured at industrial scale with appropriate consistency between individual batches. However, the data obtained using the light chain LC-MS method also demonstrates variation in the content of individual Sym001 antibodies. We are confident that future rpAb products, based on rational selection of antibodies and their individual manufacturing cell lines, will allow the manufacturing of such products with optimized and much more even distribution of the comprised antibodies.18 Such future product candidates will be developed on the basis of a careful selection of constituent antibodies based on a variety of characteristics including antigen-binding reactivity, potency, protein chemical characteristics, and expression behavior. On the basis of the target in question, the associated antigens, and the perceived mechanism of action, future rpAb products are likely to contain fewer constituent antibodies, e.g., 5-10 antibodies. The light chain LC-MS method will be an important analytical tool in the selection, development, and release of such future products. In conclusion, we have successfully developed a new MS based method allowing the simultaneous quantification of an rpAb product containing 25 individual antibodies. Quantification of monoclonal antibodies or proteins using MS based methods have previously been described;12,19 however, a quantitative method allowing analysis of 25 individual components of an rpAb product have never been described. This new method will become a crucial member of the analytical armament for the development and quality control of future rpAb therapeutics for use in humans.

Figure 3. Linearity of individual antibody light chains run alone and spiked into a Sym001 working standard. The response in the spike curves represents the total obtained response (i.e., light chain content in working standard + spike): (a) RhD 157, (b) RhD 203, and (c) RhD 324.

ACKNOWLEDGMENT We are grateful to Sebastian Bauer and Christine LagerquistHa¨gglund for support with the light chain LC-MS method qualification and to Yvonne Berger Larsen for excellent technical assistance.

the quality control of future rpAb products. This method could, thus, become a critical method potentially allowing specifica-

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

(18) Nielsen, L. S.; Baer, A.; Muller, C.; Gregersen, K.; Monster, N. T.; Rasmussen, S. K.; Weilguny, D.; Tolstrup, A. B. Mol. Biotechnol. 2010, 45, 257–266. (19) Damen, C. W.; Rosing, H.; Schellens, J. H.; Beijnen, J. H. J. Pharm. Biomed. Anal. 2008, 46, 449–455.

Received for review May 5, 2010. Accepted July 26, 2010.

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