12 research outputs found
Advanced Process Control and Automation with Special Focus on Emerging Continuous Bioprocessing
Legacy batch processing carried out in pharmaceutical and biopharmaceutical sectors is undergoing transformation to adopt the next generation continuous processing to produce safe and effective drugs with better efficiency and consistency at a reduced cost. To facilitate innovative continuous processing, enabled by an end-to-end process with a single uninterrupted production scenario, it is essential to generate real-time or near-real-time data using process analytical technology (PAT), which has been defined by the FDA as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality by design (QbD) principles, PAT-enabled data monitoring is essential for the timely control of critical process parameters (CPPs) and critical quality attributes (CQAs) to keep the process in a desired state of control to achieve a predefined product quality. Based on QbD philosophy, quality cannot be tested into products; it should be built-in or should be by design. Deployment of PAT tools for real-time monitoring is integral to align with the guiding principles of QbD-enabled workflow to enhance process and product understanding to administer a control strategy to keep the process within the design space. Aim of this chapter is to highlight the recent advancements in PAT tool-development to monitor and control CPPs and CQAs
When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid
With the advent of new initiatives
to develop chemically defined
media, cell culture scientists screen many additives to improve cell
growth and productivity. However, the introduction or increase of
supplements, typically considered beneficial or protective on their
own, to the basal media or feed stream may cause unexpected detrimental
consequences to product quality. For instance, because cultured cells
are constantly under oxidative stress, ascorbic acid (vitamin C, a
potent natural reducing agent) is a common additive to cell culture
media. However, as reported herein, a recombinant monoclonal antibody
(adalimumab) in cell culture was covalently modified by xylosone (molecular
weight 148), an oxidative product of ascorbate. Containing reactive
carbonyl groups, xylosone modifies various amines (e.g., the N-termini
of the heavy and light chains and susceptible lysines), forming either
hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our
findings show, for the first time, that ascorbate-derived xylosone
can contribute to an increase in molecular heterogeneity, such as
acidic species. Our work serves as a reminder that additives to cell
culture and their metabolites may become reactive and negatively impact
the overall product quality and should be carefully monitored with
any changes in cell culture conditions
Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody
Recombinant therapeutic monoclonal
antibodies exhibit a high degree
of heterogeneity that can arise from various post-translational modifications.
The formulation for a protein product is to maintain a specific pH
and to minimize further modifications. Generally Recognized as Safe
(GRAS), citric acid is commonly used for formulation to maintain a
pH at a range between 3 and 6 and is generally considered chemically
inert. However, as we reported herein, citric acid covalently modified
a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered
formulation at pH 5.2 and led to the formation of so-called “acidic
species” that showed mass increases of 174 and 156 Da, respectively.
Peptide mapping revealed that the modification occurred at the N-terminus
of the light chain. Three additional antibodies also showed the same
modification but displayed different susceptibilities of the N-termini
of the light chain, heavy chain, or both. Thus, ostensibly unreactive
excipients under certain conditions may increase heterogeneity and
acidic species in formulated recombinant monoclonal antibodies. By
analogy, other molecules (e.g., succinic acid) with two or more carboxylic
acid groups and capable of forming an anhydride may exhibit similar
reactivities. Altogether, our findings again reminded us that it is
prudent to consider formulations as a potential source for chemical
modifications and product heterogeneity
Detection of Alkynes via Click Chemistry with a Brominated Coumarin Azide by Simultaneous Fluorescence and Isotopic Signatures in Mass Spectrometry
Alkynes
are a key component of click chemistry and used for a wide
variety of applications including bioconjugation, selective tagging
of protein modifications, and labeling of metabolites and drug targets.
However, challenges still exist for detecting alkynes because most
1,2,3-triazole products from alkynes and azides do not possess distinct
intrinsic properties that can be used for their facile detection by
either fluorescence or mass spectrometry. To address this critical
need, a novel brominated coumarin azide was used to tag alkynes and
detect alkyne-conjugated biomolecules. This tag has several useful
properties: first, it is fluorogenic and the click-chemistry products
are highly fluorescent and quantifiable; second, its distinct isotopic
pattern facilitates identification by mass spectrometry; and third,
its click-chemistry products form a unique pair of reporter ions upon
fragmentation that can be used for the quick screening of data. Using
a monoclonal antibody conjugated with alkynes, a general workflow
has been developed and examined comprehensively