Frank Macchi - Academia.edu (original) (raw)
Papers by Frank Macchi
Analytical Chemistry, Jul 29, 2020
Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for... more Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for patients or affect product stability, thus prompting a critical need to monitor HCPs in drug substance or product to ensure product safety and quality. Current approaches for robust HCP identification at or above 10 ppm levels require either concatenated peptide fractionation or enrichment via antibody depletion, which challenges the direct quantitation of HCPs. This paper describes a simple, fast sample preparation method without the need for sample fractionation or enrichment; instead, we utilize trypsin-friendly sodium deoxycholate (SDC) as an advantageous denaturant that can be effectively removed following acidification at the end of sample digestion. This new approach enables the end-to-end onedimensional liquid chromatography−tandem mass spectrometry (1D LC−MS/MS) workflow (i.e., from sample preparation to HCP identification) to be completed in 7−8 h while demonstrating the ability to consistently identify HCPs across a broad molecular weight range at 10 ppm or above.
Journal of Chromatography B: Biomedical Sciences and Applications, Mar 1, 2001
Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were r... more Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.
Journal of Pharmacological and Toxicological Methods, Nov 1, 2018
Introduction: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibrob... more Introduction: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoβ (KLB) half-1 S Rajan and J Sonoda contributed equally to this work
Humana Press eBooks, Nov 14, 2003
Journal of pharmacological and toxicological methods, Jan 13, 2018
bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Fa... more bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoβ (KLB) half-antibody, using the knobs-into-holes strategy. bFKB1 acts as a highly selective agonist for the FGFR1/KLB receptor complex and is intended to ameliorate obesity-associated metabolic defects by mimicking the activity of the hormone FGF21. An important aspect of the biologics product manufacturing process is to establish meaningful product specifications regarding the tolerable levels of impurities that copurify with the drug product. The aim of the current study was to determine acceptable levels of product-related impurities for bFKB1. To determine the tolerable levels of these impurities, we dosed obese mice with bFKB1 enriched with various levels of either HMW impurities or anti-FGFR1-related impurities, and measured biomarkers for KLB-independent FGFR1 signaling. Here, we show that product-related impurities of bFKB1,...
Journal of Chromatography B: Biomedical Sciences and Applications, 2001
Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were r... more Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.
Biotechnology and bioengineering, Oct 14, 2016
Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is ... more Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a ...
Journal of Pharmaceutical and Biomedical Analysis
Analytical chemistry, Jan 16, 2015
Accurate and precise quantitative measurement of product-related variants of a therapeutic antibo... more Accurate and precise quantitative measurement of product-related variants of a therapeutic antibody is essential for product development and testing. Bispecific antibodies (bsAbs) are Abs composed of two different half-antibody arms, each of which recognizes a distinct target, and recently they have attracted substantial therapeutic interest. Due to the increased complexity of its structure and its production process, as compared to a conventional monoclonal antibody, additional product-related variants, including covalent and non-covalent homodimers of half antibodies (hAbs), may be present in the bsAb product. Sufficient separation and reliable quantitation of these bsAb homodimers using liquid chromatography (LC) or capillary electrophoresis-based methods is challenging because these homodimer species and the bsAb often have similar physicochemical properties. Formation of non-covalent homodimers and heterodimers can also occur. In addition, since homodimers share common sequence...
Methods in molecular biology (Clifton, N.J.), 2000
Current Trends in Monoclonal Antibody Development and Manufacturing, 2009
... Reed J. Harris, Edward T. Chin, Frank Macchi, Rodney G. Keck, Bao-Jen Shyong, Victor T. Ling,... more ... Reed J. Harris, Edward T. Chin, Frank Macchi, Rodney G. Keck, Bao-Jen Shyong, Victor T. Ling, Armando J. Cordoba, Melinda Marian, Don Sinclair ... Proc Natl Acad Sci USA 93:55125516 Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka H, Iida S ...
Molecular Pharmaceutics, 2013
The reaction of singlet oxygen with water to form hydrogen peroxide was catalyzed by antibodies a... more The reaction of singlet oxygen with water to form hydrogen peroxide was catalyzed by antibodies and has been termed as the antibody catalyzed water oxidation pathway (ACWOP) (Nieva and Wentworth, Trends Biochem. Sci. 2004, 29, 274−278; Nieva et al. Immunol. Lett. 2006, 103, 33−38). While conserved and buried tryptophans in the antibody are thought to play a major role in this pathway, our studies with a monoclonal antibody, mAb-1 and its mutant W53A, clearly demonstrate the role of surface-exposed tryptophans in production of hydrogen peroxide, via the photo-oxidation pathway. Reactive oxygen species (ROS) such as singlet oxygen and superoxide were detected and site-specific tryptophan (Trp53) oxidation was observed under these conditions using RP-HPLC and mass spectrometry. The single mutant of the surface exposed Trp53 to Ala53 (W53A) results in a 50% reduction in hydrogen peroxide generated under these conditions, indicating that surface exposed tryptophans are highly efficient in transferring light energy to oxygen and contribute significantly to ROS generation. ACWOP potentially leads to the chemical instability of mAb-1 via the generation of ROS and is important to consider during clinical and pharmaceutical development of mAbs.
Journal of Chromatography B: Biomedical Sciences and Applications, 2001
Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were r... more Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions. H monoclonal antibodies whose light chain and heavy glycosylation differences that influence F effector c chain sequences are known in advance of any functions [5], or antibody fragmentation [6]. Assays chemical characterization, there would appear to be for the determination of the extent of overall deamidation [7], the types of N-linked oligosaccharides [8], or the extent of heavy chain glycosylation [9] are *Corresponding author. Tel.: 11-650-225-4187; fax: 11-650available. The processing of C-terminal lysine res-225-3554.
Analytical Chemistry, Jul 29, 2020
Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for... more Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for patients or affect product stability, thus prompting a critical need to monitor HCPs in drug substance or product to ensure product safety and quality. Current approaches for robust HCP identification at or above 10 ppm levels require either concatenated peptide fractionation or enrichment via antibody depletion, which challenges the direct quantitation of HCPs. This paper describes a simple, fast sample preparation method without the need for sample fractionation or enrichment; instead, we utilize trypsin-friendly sodium deoxycholate (SDC) as an advantageous denaturant that can be effectively removed following acidification at the end of sample digestion. This new approach enables the end-to-end onedimensional liquid chromatography−tandem mass spectrometry (1D LC−MS/MS) workflow (i.e., from sample preparation to HCP identification) to be completed in 7−8 h while demonstrating the ability to consistently identify HCPs across a broad molecular weight range at 10 ppm or above.
Journal of Chromatography B: Biomedical Sciences and Applications, Mar 1, 2001
Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were r... more Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.
Journal of Pharmacological and Toxicological Methods, Nov 1, 2018
Introduction: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibrob... more Introduction: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoβ (KLB) half-1 S Rajan and J Sonoda contributed equally to this work
Humana Press eBooks, Nov 14, 2003
Journal of pharmacological and toxicological methods, Jan 13, 2018
bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Fa... more bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoβ (KLB) half-antibody, using the knobs-into-holes strategy. bFKB1 acts as a highly selective agonist for the FGFR1/KLB receptor complex and is intended to ameliorate obesity-associated metabolic defects by mimicking the activity of the hormone FGF21. An important aspect of the biologics product manufacturing process is to establish meaningful product specifications regarding the tolerable levels of impurities that copurify with the drug product. The aim of the current study was to determine acceptable levels of product-related impurities for bFKB1. To determine the tolerable levels of these impurities, we dosed obese mice with bFKB1 enriched with various levels of either HMW impurities or anti-FGFR1-related impurities, and measured biomarkers for KLB-independent FGFR1 signaling. Here, we show that product-related impurities of bFKB1,...
Journal of Chromatography B: Biomedical Sciences and Applications, 2001
Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were r... more Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.
Biotechnology and bioengineering, Oct 14, 2016
Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is ... more Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a ...
Journal of Pharmaceutical and Biomedical Analysis
Analytical chemistry, Jan 16, 2015
Accurate and precise quantitative measurement of product-related variants of a therapeutic antibo... more Accurate and precise quantitative measurement of product-related variants of a therapeutic antibody is essential for product development and testing. Bispecific antibodies (bsAbs) are Abs composed of two different half-antibody arms, each of which recognizes a distinct target, and recently they have attracted substantial therapeutic interest. Due to the increased complexity of its structure and its production process, as compared to a conventional monoclonal antibody, additional product-related variants, including covalent and non-covalent homodimers of half antibodies (hAbs), may be present in the bsAb product. Sufficient separation and reliable quantitation of these bsAb homodimers using liquid chromatography (LC) or capillary electrophoresis-based methods is challenging because these homodimer species and the bsAb often have similar physicochemical properties. Formation of non-covalent homodimers and heterodimers can also occur. In addition, since homodimers share common sequence...
Methods in molecular biology (Clifton, N.J.), 2000
Current Trends in Monoclonal Antibody Development and Manufacturing, 2009
... Reed J. Harris, Edward T. Chin, Frank Macchi, Rodney G. Keck, Bao-Jen Shyong, Victor T. Ling,... more ... Reed J. Harris, Edward T. Chin, Frank Macchi, Rodney G. Keck, Bao-Jen Shyong, Victor T. Ling, Armando J. Cordoba, Melinda Marian, Don Sinclair ... Proc Natl Acad Sci USA 93:55125516 Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka H, Iida S ...
Molecular Pharmaceutics, 2013
The reaction of singlet oxygen with water to form hydrogen peroxide was catalyzed by antibodies a... more The reaction of singlet oxygen with water to form hydrogen peroxide was catalyzed by antibodies and has been termed as the antibody catalyzed water oxidation pathway (ACWOP) (Nieva and Wentworth, Trends Biochem. Sci. 2004, 29, 274−278; Nieva et al. Immunol. Lett. 2006, 103, 33−38). While conserved and buried tryptophans in the antibody are thought to play a major role in this pathway, our studies with a monoclonal antibody, mAb-1 and its mutant W53A, clearly demonstrate the role of surface-exposed tryptophans in production of hydrogen peroxide, via the photo-oxidation pathway. Reactive oxygen species (ROS) such as singlet oxygen and superoxide were detected and site-specific tryptophan (Trp53) oxidation was observed under these conditions using RP-HPLC and mass spectrometry. The single mutant of the surface exposed Trp53 to Ala53 (W53A) results in a 50% reduction in hydrogen peroxide generated under these conditions, indicating that surface exposed tryptophans are highly efficient in transferring light energy to oxygen and contribute significantly to ROS generation. ACWOP potentially leads to the chemical instability of mAb-1 via the generation of ROS and is important to consider during clinical and pharmaceutical development of mAbs.
Journal of Chromatography B: Biomedical Sciences and Applications, 2001
Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were r... more Seven forms of a therapeutic recombinant antibody that binds to the her2 /neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions. H monoclonal antibodies whose light chain and heavy glycosylation differences that influence F effector c chain sequences are known in advance of any functions [5], or antibody fragmentation [6]. Assays chemical characterization, there would appear to be for the determination of the extent of overall deamidation [7], the types of N-linked oligosaccharides [8], or the extent of heavy chain glycosylation [9] are *Corresponding author. Tel.: 11-650-225-4187; fax: 11-650available. The processing of C-terminal lysine res-225-3554.