Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.     

Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C‐terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C‐terminal lysine (‐K) or lysine and glycine (‐GK). Interestingly, clones that express antibodies lacking HC C‐terminal lysine (‐K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (‐GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C‐terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:786–794, 2017     

EGFR: Tale of the C‐terminal tail

The carboxy terminal tail of epidermal growth factor receptor (EGFR) plays a critical role in the regulation of the enzyme activity of the kinase. There is a good structural model for the mechanism by which the C‐terminal tail proximal to the kinase domain contributes to the negative regulation of the activity. Its conformation in the active state, conversely, has remained elusive due to its dynamic nature. A recently published structure of EGFR kinase domain shows the conformation of the proximal C‐terminal tail in the active kinase. Analysis of this conformational state of the C‐terminal tail is presented, and some of the mutagenesis data is revisited. © 2013 The Protein Society     

C‐terminal domain of SARS‐CoV main protease can form a 3D domain‐swapped dimer

SARS coronavirus main protease (M^pro) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. We have reported that both the M^pro C-terminal domain alone (M^pro-C) and the N-finger deletion mutant of M^pro (M^pro-Δ7) exist as a stable dimer and a stable monomer (Zhong et al., J Virol 2008; 82:4227-4234). Here, we report structures of both M^pro-C monomer and dimer. The structure of the M^pro-C monomer is almost identical to that of the C-terminal domain in the crystal structure of M^pro. Interestingly, the M^pro-C dimer structure is characterized by 3D domain-swapping, in which the first helices of the two protomers are interchanged and each is enwrapped by four other helices from the other protomer. Each folding subunit of the M^pro-C domain-swapped dimer still has the same general fold as that of the M^pro-C monomer. This special dimerization elucidates the structural basis for the observation that there is no exchange between monomeric and dimeric forms of M^pro-C and M^pro-Δ7.     

C‐terminal domains of bacterial proteases: structure,function and the biotechnological applications

C‐terminal domains widely exist in the C‐terminal region of multidomain proteases. As a β‐sandwich domain in multidomain protease, the C‐terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C‐terminal protease domains are described. Furthermore, the application prospects of C‐terminal domains, including polycystic kidney disease, prepeptidase C‐terminal and collagen‐binding domain, in the area of medicine and biological artificial materials are also discussed.     

The structure of S. lividans acetoacetyl‐CoA synthetase shows a novel interaction between the C‐terminal extension and the N‐terminal domain

The adenosine monoposphate‐forming acyl‐CoA synthetase enzymes catalyze a two‐step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ～110 residue C‐terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl‐CoA synthetase (AacS) is presented that illustrates a novel aspect of this C‐terminal domain. Specifically, several acetyl‐ and acetoacetyl‐CoA synthetases contain a 30‐residue extension on the C‐terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N‐terminal domain. Proteins 2015; 83:575–581. © 2014 Wiley Periodicals, Inc.     

Hydrophobic benzyl amines as supports for liquid‐phase C‐terminal amidated peptide synthesis: application to the preparation of ABT‐510

C‐terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C‐terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C‐terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work‐up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag‐assisted liquid‐phase peptide synthesis as supports, leading to the total synthesis of ABT‐510 ( 2 ). Although an ethyl amide‐forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C‐terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Design,synthesis, and comparative evaluation of 99mTc(CO)3‐labeled N‐terminal and C‐terminal modified asparagine–glycine–arginine peptide constructs

The present study describes modification of asparagine–glycine–arginine (NGR) peptide at N‐terminally and C‐terminally by introduction of a tridentate chelating scaffold via click chemistry reaction. The N‐terminal and C‐terminal modified peptides were radiometalated with [^99mTc(CO)₃]⁺ precursor. The influence of these moieties at the two termini on the targeting properties of NGR peptide was determined by in vitro cell uptake studies and in vivo biodistribution studies. The two radiolabeled constructs did not exhibit any significant variation in uptake in murine melanoma B16F10 cells during in vitro studies. In vivo studies revealed nearly similar tumor uptake of N‐terminally modified peptide construct 5 and C‐terminally construct 6 at 2 h p.i. (1.9 ± 0.1 vs 2.4 ± 0.2% ID/g, respectively). The tumor‐to‐blood (T/B) and tumor‐to‐liver (T/L) ratios of the two radiometalated peptides were also quite similar. The two constructs cleared from all the major organs (heart, lungs, spleen, stomach, and blood) at 4 h p.i. (<1% ID/g). Blocking studies carried out by coinjection of cCNGRC peptide led to approximately 50% reduction in the tumor uptake at 2 h p.i. This work thus illustrates the possibility of convenient modification/radiometalation of NGR peptide at either N‐ or C‐terminus without hampering tumor targeting and pharmacokinetics.     

C‐terminal tail derived from the neighboring subunit is critical for the activity of Thermoplasma acidophilumD‐aldohexose dehydrogenase

The D ‐aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D ‐aldohexoses, especially D ‐mannose. AldT comprises a unique C‐terminal tail motif (residues 247–255) that shuts the active‐site pocket of the neighboring subunit. The functional role of the C‐terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C‐terminal deletion mutants (Δ254, Δ253, Δ252, and Δ249) and two site‐specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C‐terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Δ249 mutant was also determined. The structure showed that the active‐site loops undergo a significant conformational change, which leads to the structural deformation of the substrate‐binding pocket. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A special reactor design for investigations of mixing time effects in a scaled‐down industrial L‐lysine fed‐batch fermentation process

A specially designed model reactor based on a 42‐L laboratory fermentor was equipped with six stirrers (Rushton turbines) and five cylindrical disks. In this model reactor, the mixing time, Θ₉₀, turned out to be 13 times longer compared with the 42‐L standard laboratory fermentor fitted with two Rushton turbines and four wall‐fixed longitudinal baffles. To prove the suitability of the model reactor for scaledown studies of mixing‐time‐dependent processes, parallel exponential fed‐batch cultivations were carried out with the leucine‐auxotrophic strain, Corynebacterium glutamicum DSM 5715, serving as a microbial test system. L‐ Leucine, the process‐limiting substrate, was fed onto the liquid surface of both reactors. Cultivations were conducted using the same inoculum material and equal oxygen supply. The model reactor showed reduced sugar consumption (−14%), reduced ammonium consumption (−19%), and reduced biomass formation (−7%), which resulted in a decrease in L ‐lysine formation (−12%). These findings were reflected in less specific enzyme activity, which was determined for citrate synthase (CS), phosphoenolpyruvate carboxylase (PEP‐C), and aspartate kinase (AK). The reduced specific activity of CS correlated with lower CO₂ evolution (−36%) during cultivation. The model reactor represents a valuable tool to simulate the conditions of poor mixing and inhomogeneous substrate distribution in bioreactors of industrial scale. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 599–606, 1999.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Microtubule C‐Terminal Tails Can Change Characteristics of Motor Force Production

Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors' force production and detachment kinetics are critical for their group function, but how microtubule (MT) details affect these properties – if at all – is unknown. We investigated these questions using both a vesicular transport human kinesin, kinesin‐1, and also a mitotic kinesin likely optimized for group function, kinesin‐5, moving along either bovine brain or MCF7(breast cancer) MTs. We found that kinesin‐1 functioned similarly on the two sets of MTs – in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of kinesin‐1 force production was slightly decreased on MCF7 MTs. In contrast, kinesin‐5's function changed dramatically on MCF7 MTs: its average detachment force was reduced and its force–velocity curve was different. In spite of the reduced detachment force, the force–velocity alteration surprisingly improved high‐load group function for kinesin‐5 on the cancer‐cell MTs, potentially contributing to functions such as spindle‐mediated chromosome separation. Significant differences were previously reported for C‐terminal tubulin tails in MCF7 versus bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of MTs similar.     

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α‐position and peptides on N‐terminal position

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N‐α or ε‐acetyl‐L‐lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε‐position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N‐acylation on the α‐position of the lysine or of the amino‐acid in N‐terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N‐α‐acetyl‐L‐lysine could be attributed to the product of SAM23877_1734 gene.