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Acid hydrolysis of monoclonal antibodies
IMMuIIoLoGleAL Journalof Immunological Methcd~ 185 0995) X77-t8U
ELSNIER
METHOOS
Acid hydrolysis of monoclonal antibodies
For analysis of monoclonal antibodies using polyaaylamide gel electrophoresis, two hydrolytic fragments derived from the heavy chain of mouse IgGl were produced during incubaticm of the antibodies in Laemmli reducing sample buffer 1OWCfor 5 min. The cleavage sites were identified by amino terminal sequencing. Results indicate that the fmal pH of the mixture is critical for the production of the fragments which are generated when the pH is approximately 6.0. At pH 8.0, no fragments are detected. The relevance of this finding to
at
those working with monoclonal antibodies is discussed. Keyword:
Monoclonal
antibody; Elcctropboresis;
Acid hydrolysis; Heavy chain
1. lotmduction SDS-PAGE electrophoresis is a powerful, easy to use technique that has been employed extensively to monitor purification and stability of proteins. The Laemmli protocol (Laemmli, 1970) is the procedure most commonly followed. In this system the sample is diluted l/2 with a sample buffer, a Tris-HCI, pH 6.8 solution containing &mercaptoethanol, glycerol and bromophenol blue. The mixture is heated for 3-5 min at l!WC, and after cooling, loaded into the wells of the eltctrophoresis gel. Current investigation in our laboratory concerns the stability of several therapeutic antibodies, under normal as well as stressful conditions. After storage, these proteins are analyzed with different techniques, including SDS-PAGE, in
-Ez&cmdi”g fE1g)357-8348.
author.
Tel.:
@18)305-6099; Fax:
which a stable IgG molecule is composed of heavy (50 kDa) and light (30 kDa) chains only. In this study, we characterize other cleavage products of three IgGl mouse monoclonal antibodies resulting only from boiling the samples in Laemmli sample buffer. To eliminate this artifact, we propose changes in the classical Laemmli sample buffer to avoid the non-specific production of these fragments.
2. Materials and methods In the current study, mouse monoclonal antibody 9069 was prepared in phosphate buffet at two different pHs: 7.2 and 5.5 (formulations 1 and 2, respectively). Additional mouse monoclonal antibodies 9187 and 9189 and rat antibcdies YTH24 and YTH54 were also studied, prepared in formulation 2.
0022.1759/95/%9.50 B, 1995 Elsevier Science B.V. All rights resewed SSLJi oozt-1759(95)00110-7
Reducing SDS-PAGE protocol was conductec on precast 4-u)% actylamide gels !Novex, Encinitas, CA, cat. EC6028) with a Novex vertical electrophoresis system and Bio-Rad model 2i?O/2.0 power supply. Samples were prepared by diluting a 2 mg/tnl antibody solution with an equal volume of Novex Laemmli samplebuffer (Tris-HCI 0.125 M, glyceml 20%, SDS 4%, bromophenol blue 0.005%, pH 6.8; Novex cat. LC 2676) or alternative Novex sample buffer Lci676 (Tris-HCI 0.9 M, glycerol 24%, SDS 81, Coomake blue G 0.0075%. Phenol Red 0.0025%, pH 8.45) containing 10% 2-mercaptoethanol. The diluted samples were then boiled at 100°C for 5 min. The pH of the sample/buffer mixtures were determined using a Horiba Cardy pH meter. Each well of the gel was loaded with either 20 pg of sample protein or 10 p1 of Novex wide range moIecular weight standards. lo order to sequence the degradation bands. an optimum concentration of protein is needed. This was obtained by incubating the antibody samples at pH 2.6-2.8 at 37°C for 4-5 days, followed by reducing electmphoresis. The gel was ekctmphore.ticalIy blotted onto PVDF membrane (Towbin et al., 19791. Bands were visualized by staining with 0.1% Coomassie blue and excised. These bands were placed in a 477 Micmsequettce analysis system (Applied B&systems) and sequenced using the pulse-liquid method.
123
45
200-
an equal vohtme of an antibody solution are shown in Tabk 1. When a sample at pH 7.2 was mixed with the sample buffer, the resultant pH was 7.0. However. when the OH 5.5 samole was added to the sat&e buffer, the pH of tire mixture dropped to 6.0. Since the rate of pH change with temperature (dpH/dT) for Triv is -0.028 (Dawson et al., 1986) the predicted pH of the
3. Results The stability of the antibody solutions used in the stady was assessed at a range of temperatures from -70°C to 35°C. When an SDS-PAGE reducing gel was performed on the antibody solotions, two additional bands were observed migrating just above and below the light chain band (Fig. 1, lanes 2 and 3). These bands were more intense in the profile of the pH 5.5 sample (lane 3). Since the intensity of these two bands did not increase with increased storage temperature or time, the possibility that the bands were an artifact of the SDS-PAGE orotocol was considered. The results of pH m;asurement at 25°C of a mixture of J.aemmli sample buffer LC 2676 and
Formulalion 2+ LC2676 LC1676 Formulation I +LC1676 Fommlation ?.+ LC1676
6.0 8.4 8.1 8.L
3.9 6.2 6.0 6.0
Sample buffss mlin Trir fdpH/dT = -0.028). ’ Formulations 1 and 2 contain phosphate buffer (dpH/dT = -0.wm
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sample at 1OLPCshould be 4.9 for the high pH sample and 3.9 for the low pH sample. When the pH 5.5 and pH 7.2 formulated antibodies were mixed with sample buffer L.C 1676, the resultant pH was 8.1 for both samples. The predicted pH at 1UO”Cwas calculated to be 6.0. SDS-PAGE profile of the low and high pH formulations prepared with the new sample buffer is shown in Fig. 1, lanes 4 and 5. Note that only two bands are observed, corresponding to the heavy and light chains with no degradation product!& The SDS-PAGE profiles of two other moose antibodies and a rat antibody are shown in Fig. 2. Mouse antibody samples prepared with the Laemmli sample buffer (LC2676) show the two degradation bands, while those prepared with
LC1676 do not. The rat antibody shows no degradation products, regardless of the sample buffer used. The amino acid sequence of the lower degradation band of the three mouse antibodies is show in Table 2. The sequence is identical for all antibodies and corresponds to the constant reeion of the heaw chain. in the CH2 domain. It is noteworthy that ia each’instance the first amino acid residue in the sequence correspwds to that of proline. The amino acid sequence of the upper degradation band for two of the three murine antibodies is shown in Table 3. Tbe sequence is homologous to the amino terminal (variable) region of the heavy chain. It was not possible to determine the sequence of this band in the 9187 antibody sample because of apparent blockage of the amino terminal.
4. Discussion In the present study it was demonstrated that standard SDS-PAGE sample preparation techniques created two hydrolytic fragments of the heaw chain of three mouse leG1 antibodies (Fie. 1 anh Fig. 2). The sequence-of the upper dti> corresponded to the amino terminal of the heavy chain (Table 3). The lower band has a sequence that begins at amino acid 283 of the CH2 domain of the heavy chain (Table 2) &hat et al., 1991). An aspartic acid residue precedes a proline according to both the sequence of the gene for the 9069 antibody and the consensus sequence for IgGl murine heavy chain (Kabat et al., 1991).
All three mouse IgGl antibodies studied cleaved in the same manner, since they all share the same constant region in the heavy chain. Another antibody, a rat IgG2, did not cleave under the same ccmditions, due to the fact that there is a glutamic acid residue instcad of an aspartic at amino acid 282 of the CH2 domain of the heay chain. The mechanism of hydrolysis is mat likely nucleophilic attack of the carboxy group of aspartic acid over the carbonyl group of the peptidc bond between asp&c acid and proline. Two factors contribute to the special lability of this bond. Fist, in the free amino acid state. the nitrogen in the imido ring of praline has a pKa of 10.6. mere basic than the nitrogen in the amino groups of other free amino acids, which range in pKa from 8.4 to 9.9 (Dawson et al., 1986). This basic character may contribute to a weakening of the peptide bond between asparty and prolyl residues. Secondly, the carboxyl group of aspartic acid is necessary for nucleophilic attack of the carbonyl group of the peptide bond between aspartic and pmline (Piszkiewicz et al.. 1970). This reaction also requires favorable steric environment, e.g. the glutamic-praline peptide bond does not exhibit this lability because of the difference in side chain length between the aspartic residue and the glutamic residue. Changing the classic Laemmli sample buffer to a new sample buffer with a higher tris concentra-
tion
and a pH of 8.4 is recommended when performing SDS-PAGE of mouse IgGl, or any other protein containing an aspartic-proline bond. Preparation techniques should be meticulously evaluated for their suitability. In this case, the buffering cwacity of a commonly used buffer was inadequate for the evaluation of acidic samples. In addition. misintemretation of the mofile obtained throw& SDSIPAGE can be avoided by careful evaluation of sample preparation techniques.
ELSNIER
METHOOS
Acid hydrolysis of monoclonal antibodies
For analysis of monoclonal antibodies using polyaaylamide gel electrophoresis, two hydrolytic fragments derived from the heavy chain of mouse IgGl were produced during incubaticm of the antibodies in Laemmli reducing sample buffer 1OWCfor 5 min. The cleavage sites were identified by amino terminal sequencing. Results indicate that the fmal pH of the mixture is critical for the production of the fragments which are generated when the pH is approximately 6.0. At pH 8.0, no fragments are detected. The relevance of this finding to
at
those working with monoclonal antibodies is discussed. Keyword:
Monoclonal
antibody; Elcctropboresis;
Acid hydrolysis; Heavy chain
1. lotmduction SDS-PAGE electrophoresis is a powerful, easy to use technique that has been employed extensively to monitor purification and stability of proteins. The Laemmli protocol (Laemmli, 1970) is the procedure most commonly followed. In this system the sample is diluted l/2 with a sample buffer, a Tris-HCI, pH 6.8 solution containing &mercaptoethanol, glycerol and bromophenol blue. The mixture is heated for 3-5 min at l!WC, and after cooling, loaded into the wells of the eltctrophoresis gel. Current investigation in our laboratory concerns the stability of several therapeutic antibodies, under normal as well as stressful conditions. After storage, these proteins are analyzed with different techniques, including SDS-PAGE, in
-Ez&cmdi”g fE1g)357-8348.
author.
Tel.:
@18)305-6099; Fax:
which a stable IgG molecule is composed of heavy (50 kDa) and light (30 kDa) chains only. In this study, we characterize other cleavage products of three IgGl mouse monoclonal antibodies resulting only from boiling the samples in Laemmli sample buffer. To eliminate this artifact, we propose changes in the classical Laemmli sample buffer to avoid the non-specific production of these fragments.
2. Materials and methods In the current study, mouse monoclonal antibody 9069 was prepared in phosphate buffet at two different pHs: 7.2 and 5.5 (formulations 1 and 2, respectively). Additional mouse monoclonal antibodies 9187 and 9189 and rat antibcdies YTH24 and YTH54 were also studied, prepared in formulation 2.
0022.1759/95/%9.50 B, 1995 Elsevier Science B.V. All rights resewed SSLJi oozt-1759(95)00110-7
Reducing SDS-PAGE protocol was conductec on precast 4-u)% actylamide gels !Novex, Encinitas, CA, cat. EC6028) with a Novex vertical electrophoresis system and Bio-Rad model 2i?O/2.0 power supply. Samples were prepared by diluting a 2 mg/tnl antibody solution with an equal volume of Novex Laemmli samplebuffer (Tris-HCI 0.125 M, glyceml 20%, SDS 4%, bromophenol blue 0.005%, pH 6.8; Novex cat. LC 2676) or alternative Novex sample buffer Lci676 (Tris-HCI 0.9 M, glycerol 24%, SDS 81, Coomake blue G 0.0075%. Phenol Red 0.0025%, pH 8.45) containing 10% 2-mercaptoethanol. The diluted samples were then boiled at 100°C for 5 min. The pH of the sample/buffer mixtures were determined using a Horiba Cardy pH meter. Each well of the gel was loaded with either 20 pg of sample protein or 10 p1 of Novex wide range moIecular weight standards. lo order to sequence the degradation bands. an optimum concentration of protein is needed. This was obtained by incubating the antibody samples at pH 2.6-2.8 at 37°C for 4-5 days, followed by reducing electmphoresis. The gel was ekctmphore.ticalIy blotted onto PVDF membrane (Towbin et al., 19791. Bands were visualized by staining with 0.1% Coomassie blue and excised. These bands were placed in a 477 Micmsequettce analysis system (Applied B&systems) and sequenced using the pulse-liquid method.
123
45
200-
an equal vohtme of an antibody solution are shown in Tabk 1. When a sample at pH 7.2 was mixed with the sample buffer, the resultant pH was 7.0. However. when the OH 5.5 samole was added to the sat&e buffer, the pH of tire mixture dropped to 6.0. Since the rate of pH change with temperature (dpH/dT) for Triv is -0.028 (Dawson et al., 1986) the predicted pH of the
3. Results The stability of the antibody solutions used in the stady was assessed at a range of temperatures from -70°C to 35°C. When an SDS-PAGE reducing gel was performed on the antibody solotions, two additional bands were observed migrating just above and below the light chain band (Fig. 1, lanes 2 and 3). These bands were more intense in the profile of the pH 5.5 sample (lane 3). Since the intensity of these two bands did not increase with increased storage temperature or time, the possibility that the bands were an artifact of the SDS-PAGE orotocol was considered. The results of pH m;asurement at 25°C of a mixture of J.aemmli sample buffer LC 2676 and
Formulalion 2+ LC2676 LC1676 Formulation I +LC1676 Fommlation ?.+ LC1676
6.0 8.4 8.1 8.L
3.9 6.2 6.0 6.0
Sample buffss mlin Trir fdpH/dT = -0.028). ’ Formulations 1 and 2 contain phosphate buffer (dpH/dT = -0.wm
12345678910
sample at 1OLPCshould be 4.9 for the high pH sample and 3.9 for the low pH sample. When the pH 5.5 and pH 7.2 formulated antibodies were mixed with sample buffer L.C 1676, the resultant pH was 8.1 for both samples. The predicted pH at 1UO”Cwas calculated to be 6.0. SDS-PAGE profile of the low and high pH formulations prepared with the new sample buffer is shown in Fig. 1, lanes 4 and 5. Note that only two bands are observed, corresponding to the heavy and light chains with no degradation product!& The SDS-PAGE profiles of two other moose antibodies and a rat antibody are shown in Fig. 2. Mouse antibody samples prepared with the Laemmli sample buffer (LC2676) show the two degradation bands, while those prepared with
LC1676 do not. The rat antibody shows no degradation products, regardless of the sample buffer used. The amino acid sequence of the lower degradation band of the three mouse antibodies is show in Table 2. The sequence is identical for all antibodies and corresponds to the constant reeion of the heaw chain. in the CH2 domain. It is noteworthy that ia each’instance the first amino acid residue in the sequence correspwds to that of proline. The amino acid sequence of the upper degradation band for two of the three murine antibodies is shown in Table 3. Tbe sequence is homologous to the amino terminal (variable) region of the heavy chain. It was not possible to determine the sequence of this band in the 9187 antibody sample because of apparent blockage of the amino terminal.
4. Discussion In the present study it was demonstrated that standard SDS-PAGE sample preparation techniques created two hydrolytic fragments of the heaw chain of three mouse leG1 antibodies (Fie. 1 anh Fig. 2). The sequence-of the upper dti> corresponded to the amino terminal of the heavy chain (Table 3). The lower band has a sequence that begins at amino acid 283 of the CH2 domain of the heavy chain (Table 2) &hat et al., 1991). An aspartic acid residue precedes a proline according to both the sequence of the gene for the 9069 antibody and the consensus sequence for IgGl murine heavy chain (Kabat et al., 1991).
All three mouse IgGl antibodies studied cleaved in the same manner, since they all share the same constant region in the heavy chain. Another antibody, a rat IgG2, did not cleave under the same ccmditions, due to the fact that there is a glutamic acid residue instcad of an aspartic at amino acid 282 of the CH2 domain of the heay chain. The mechanism of hydrolysis is mat likely nucleophilic attack of the carboxy group of aspartic acid over the carbonyl group of the peptidc bond between asp&c acid and proline. Two factors contribute to the special lability of this bond. Fist, in the free amino acid state. the nitrogen in the imido ring of praline has a pKa of 10.6. mere basic than the nitrogen in the amino groups of other free amino acids, which range in pKa from 8.4 to 9.9 (Dawson et al., 1986). This basic character may contribute to a weakening of the peptide bond between asparty and prolyl residues. Secondly, the carboxyl group of aspartic acid is necessary for nucleophilic attack of the carbonyl group of the peptide bond between aspartic and pmline (Piszkiewicz et al.. 1970). This reaction also requires favorable steric environment, e.g. the glutamic-praline peptide bond does not exhibit this lability because of the difference in side chain length between the aspartic residue and the glutamic residue. Changing the classic Laemmli sample buffer to a new sample buffer with a higher tris concentra-
tion
and a pH of 8.4 is recommended when performing SDS-PAGE of mouse IgGl, or any other protein containing an aspartic-proline bond. Preparation techniques should be meticulously evaluated for their suitability. In this case, the buffering cwacity of a commonly used buffer was inadequate for the evaluation of acidic samples. In addition. misintemretation of the mofile obtained throw& SDSIPAGE can be avoided by careful evaluation of sample preparation techniques.