Alavattam Sreedhara

Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

Establishing a Link Between Amino Acid Sequences and Self-Associating and Viscoelastic Behavior of Two Closely Related Monoclonal Antibodies

ABSTRACTPurposeTo investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2.MethodsSeveral mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates. Dynamic light scattering quantified intermolecular interactions in dilute solutions; sedimentation equilibrium analysis determined the corrected weight average molecular weight (Mwc) to assess the self-associating behavior in high concentration. The molecular charge was estimated from electrophoretic mobility measurements.ResultsReplacing the charged residues in the CDR of MAb1 resulted in a lower Mwc and solution viscosity. The corresponding changes in either just the variable light (VL) or variable heavy (VH) chain showed only a partial decrease in viscosity, whereas changes in both VL and VH chains resulted in a dramatic reduction in viscosity. The converse case where the VL and VH chains of MAb2 were made to look like MAb1 did not self-associate or show increased viscosity.ConclusionsExposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.

A new roadmap for biopharmaceutical drug product development: Integrating development, validation, and quality by design

Quality by design (QbD) is a science- and risk-based approach to drug product development. Although pharmaceutical companies have historically used many of the same principles during development, this knowledge was not always formally captured or proactively submitted to regulators. In recent years, the US Food and Drug Administration has also recognized the need for more controls in the drug manufacturing processes, especially for biological therapeutics, and it has recently launched an initiative for Pharmaceutical Quality for the 21st Century to modernize pharmaceutical manufacturing and improve product quality. In the biopharmaceutical world, the QbD efforts have been mainly focused on active pharmaceutical ingredient processes with little emphasis on drug product development. We present a systematic approach to biopharmaceutical drug product development using a monoclonal antibody as an example. The approach presented herein leverages scientific understanding of products and processes, risk assessments, and rational experimental design to deliver processes that are consistent with QbD philosophy without excessive incremental effort. Data generated using these approaches will not only strengthen data packages to support specifications and manufacturing ranges but hopefully simplify implementation of postapproval changes. We anticipate that this approach will positively impact cost for companies, regulatory agencies, and patients, alike.

Characterization of the Isomerization Products of Aspartate Residues at Two Different Sites in a Monoclonal Antibody

ABSTRACTPurposeTo identify and understand isomerization products and degradation profile of different aspartate residues in an IgG1 monoclonal antibody.MethodsRecombinant IgG1 was incubated for extended periods of time in a formulation buffer at recommended and accelerated storage temperatures. Isomerization reaction products were analyzed using ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), peptide mapping, and LC-MS. Model peptides with sequences containing specific aspartate residues in IgG1 were synthesized and incubated under accelerated conditions. Products of isomerization reactions of peptides were analyzed by reverse phase chromatography (RP-HPLC) and LC-MS. X-ray crystallography data from Fab of IgG1 were used to understand mechanism of isomerization reactions.ResultsA MAb containing labile Asp32-Gly sequence in CDR I region undergoes rapid isomerization reaction and leads to formation of isoaspartate (IsoAsp) and cyclic imide (Asu) forms. Isomerization of aspartate residues was observed in a non-CDR region containing Asp74-Ser sequence. Isomerization reaction at Asp74-Ser led to formation of Asu74 and trace isoAsp74. While isoAsp32 increased linearly with time, isoAsp74 did not increase during storage. Asu32 and Asu74 followed non-linear degradation kinetics and reached steady state over time. Isomerization reaction of two different model peptides containing Asp32-Gly or Asp74-Ser with neighboring amino acid sequences as those found in the MAb result in formation of IsoAsp.ConclusionsObserved levels of Asu and trace IsoAsp at the Asp74 site are unusual for typical isomerization reactions. In addition to primary sequences, pKa, solvent exposure and high order structure around aspartate residues may have influenced isomerization reaction at Asp74 in MAbI. Different degradation profiles from the two Asp residues can influence shelf life and should be carefully evaluated during product development.

Role of surface exposed tryptophan as substrate generators for the antibody catalyzed water oxidation pathway.

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.

Effect of ambient light on monoclonal antibody product quality during small-scale mammalian cell culture process in clear glass bioreactors.

During a small‐scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small‐scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.

Compatibility and stability of pertuzumab and trastuzumab admixtures in i.v. infusion bags for coadministration.

The physical/chemical stability and potential interactions after diluting two immunoglobulin G1 monoclonal antibodies (mAb), pertuzumab (Perjeta®) and trastuzumab (Herceptin®), in a single intravenous (i.v.) infusion bag containing 0.9% saline (NaCl) solution was evaluated. As commercial products, pertuzumab and trastuzumab are administered through i.v. infusion to patients sequentially, that is, one drug after the other. To increase convenience and minimize the in-clinic time for patients, the compatibility of coadministering pertuzumab (420 and 840 mg) mixed with either 420 or 720 mg trastuzumab, respectively, in a single 250 mL polyolefin or polyvinyl chloride i.v. bag stored for up to 24 h at 5°C or 30°C was determined. The controls (i.e., pertuzumab alone in an i.v. bag, trastuzumab alone in an i.v. bag) and the mAb mixture were assessed using color, appearance, and clarity, concentration and turbidity by ultraviolet spectroscopy, particulate analysis by light obscuration, size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate, analytical ultracentrifugation, and ion-exchange chromatography. Additionally, capillary zone electrophoresis, imaged capillary isoelectric focusing, and potency were utilized to measure the stability of the admixtures containing 1:1 mixtures of pertuzumab/trastuzumab and their respective controls (420 mg pertuzumab alone and 420 mg trastuzumab alone). No observable differences were detected by the above methods in the pertuzumab/trastuzumab mixtures stored up to 24 h at either 5°C or 30°C. The physicochemical methods as listed above were able to detect both molecules as well as the minor variants in the drug mixture, even though some overlap of mAb species were seen in the chromatograms and electropherograms. Furthermore, biophysical analysis also did not show any interactions between the two mAbs or any physical instability under these conditions. Additionally, the drug mixture tested by the pertuzumab-specific inhibition of cell proliferation bioassay showed comparable potency before and after storage. On the basis of these results, pertuzumab and trastuzumab admixture in a single i.v. bag is physically and chemically stable for up to 24 h at 5°C or 30°C and can be used for clinical administration.

Effect of ambient light on IgG1 monoclonal antibodies during drug product processing and development

Photostability studies are standard stress testing conducted during drug product development of various pharmaceutical compounds, including small molecules and proteins. These studies as recommended by ICH Q1B are carried out using no less than 1.2× 10(6)lux-hours in the visible region and no less than 200Wh/m(2) in UV light. However, normal drug product processing is carried out under fluorescent lamps that emit white light almost exclusively in the >400nm region with a small UV quotient. We term these as ambient or mild light conditions. We tested several IgG1 monoclonal antibodies (mAbs 1-5) under these ambient light conditions and compared them to the ICH light conditions. All the mAbs were significantly degraded under the ICH light but several mAbs (mAbs 3-5) were processed without impacting any product quality attributes under ambient or mild light conditions. Interestingly we observed site-specific Trp oxidation in mAb1, while higher aggregation and color change were observed for mAb2 under mild light conditions. The recommended ICH light conditions have a high UV component and hence may not help to rank order photosensitivity under normal protein DP processing conditions.

Screening methods to identify indole derivatives that protect against reactive oxygen species induced tryptophan oxidation in proteins.

Oxidative damage to proteins is one of the most prominent chemical degradation pathways that are of concern for drug product development in the biotechnology industry. Especially susceptible to oxidation are the Met and Trp residues in proteins. While L-Met and L-Trp have been shown to act as antioxidants typically protecting proteins against Met and Trp oxidation, respectively, l-Trp has been shown to be particularly sensitive to light, thereby producing various reactive oxygen species (ROS), including H2O2. There is hence a need to identify nonphotosensitive molecules that can protect Trp oxidation in proteins so that they can be easily handled under drug product manufacturing conditions. A combination of screening methods, namely, cyclic voltammetry (CV) and hydrogen peroxide generation upon photoirradiation, was used to screen several molecules to identify compounds that can act as antioxidants. Specifically, indole and tryptophan with hydroxy groups on the six-membered aromatic ring were found to have lower oxidation potentials than the parent compounds and produced the least amount of H2O2 upon light exposure. These derivatives were also found to sufficiently protect tryptophan oxidation in mAb1 against a variety of reactive oxygen species such as alkyl peroxides, hydroxyl radicals, and singlet oxygen and may be useful as part of the formulation toolkit to protect against protein degradation via oxidation.

Metal ion interactions with mAbs: Part 1

Fragmentation in the hinge region of an IgG1 monoclonal antibody (mAb) can affect product stability, potentially causing changes in potency and efficacy. Metals ions, such as Cu2+, can bind to the mAb and undergo hydrolysis or oxidation, which can lead to cleavage of the molecule. To better understand the mechanism of Cu2+-mediated mAb fragmentation, hinge region cleavage products and their rates of formation were studied as a function of pH with and without Cu2+. More detailed analysis of the chemical changes was investigated using model linear and cyclic peptides (with the sequence of SCDKTHTC) derived from the upper hinge region of the mAb. Cu2+ mediated fragmentation was determined to be predominantly via a hydrolytic pathway in solution. The sites and products of hydrolytic cleavage are pH and strain dependent. In more acidic environments, rates of Cu2+ induced hinge fragmentation are significantly slower than at higher pH. Although the degradation reaction rates between the linear and cyclic peptides are not significantly different, the products of degradation vary. mAb fragmentation can be reduced by modifying His, which is a potential metal binding site and a known ligand in other metalloproteins. These results suggest that a charge may contribute to stabilization of a specific molecular structure involved in hydrolysis, leading to the possible formation of a copper binding pocket that causes increased susceptibility of the hinge region to degradation.