8 L A B O R AT O RY Therapeutic proteins at peak among pharmaceuticals Sports drug testing gains powerful aids Due to the scientific and technical developments in recombinant DNA technology and protein engineer- ing since the early 1980s, therapeutic proteins have emerged as one of the most important classes of new pharmaceuticals. Currently, more than 200 protein and peptide based drugs have gained approval by the USA’s Food and Drug Administration (FDA) and many more are under preclinical or clinical investigation. Report: Katja Walpurgis & Mario Thevis These comprise recombinant versions of natural proteins (e.g. growth fac- tors, enzymes, anticoagulants) as well as engineered proteins, protein conjugates, Fc fusion proteins, and recombinant therapeutic antibodies. Both protein re-design and chemical modifications such as glycosylation and PEGylation are strategies that can be employed to extend the serum half-life and improve the effector functions of a protein drug. Moreover, the construction of Fc fusion proteins through the attachment of an immu- noglobulin G1 (IgG1) Fc domain to a protein or peptide can provide IgG- like properties, such as a long serum half-life, due to recycling through the neonatal Fc receptor (FcRn) and a slower renal clearance. Therapeutic antibodies are comparably stable mol- ecules, which bind their targets with high affinity and specificity. Therefore, they constitute the largest and fastest growing class of protein drugs. At pre- sent, more than 50 chimeric (approx. 70% human sequences), humanised (85-90% human sequences), and fully human antibodies are approved for the treatment of autoimmune diseas- es, cancer, and other disorders. Both the approved and investi- gational protein/peptide therapeutics Indirect vs. direct Western blats for the detection of therapeutic proteins in sports drug testing. In indirect assays (A), a protein drug is detected on the basis of its properties (e. g. affinity for a TGF-ß cytokine and presence of a human Fc domain. In direct assays (B), the compound is bound by a highly specific primary antibody, which can subsequently be detected by a species-directed secondary antibody. Dr Katja Walpurgis studied Biology at the University of Bonn. Since gaining her PhD in 2013, she works as a postdoctoral researcher at the Centre for Preventive Doping Research/Institute of Biochemistry of the German Sport University Cologne. Her work focuses on the development of novel detection methods for performance- enhancing protein drugs by using different proteomics techniques, such as gel electro- phoresis, western blotting, and high resolu- tion accurate mass LC-MS. Dr Mario Thevis graduated in organic chemistry and sports sciences in 1998. He gained his PhD in Biochemistry in 2001 and did post-doctoral research at the Department of Chemistry and Biochemistry at the University of California Los Angeles (UCLA) in 2002. After being a senior researcher (2003- 2005) he became Professor for Preventive Doping Research at the German Sport University Cologne in 2006. In August 2017, he accepted the position of Director of the Institute of Biochemistry of the German Sport University in Cologne. comprise several drug candidates with potential performance-enhanc- ing properties, whose misuse in sports is restricted under the terms of the World Anti-Doping Code (WADC). While protein drugs such as eryth- ropoietin and its derivatives, human growth hormone (hGH), and human chorionic gonadotropin (hCG) can be detected by using immunological approaches such as Western blot- ting and luminescent immunoassays (LIAs), liquid chromatography and high-resolution mass spectrometry (LC-HRMS) are routinely used to test doping control samples for the pres- ence of peptidic compounds such as insulins and growth hormone releas- ing peptides (GHRPs). The proactive development of spe- cific and sensitive detection methods for emerging protein/peptide thera- peutics still missing clinical approval is the main task of preventive doping research. Many of the protein-based drugs currently undergoing clinical investigation have the same thera- peutic target: TGF- cytokines such as myostatin, activin A, and GDF-11. While myostatin is considered as the key negative regulator of skel- etal muscle mass, both GDF-11 and activin A were found to inhibit late- stage erythropoiesis. Additionally, activin A is involved in the regulation of bone formation and recent studies suggest that the cytokine could also play a role in muscle growth inhibi- tion. TGF- cytokines exert their bio- The Heraeus Symposium at DKOU The challenge of Periprosthetic infection Report: Beate Wagner joint infection (PJI) Periprosthetic is on the increase internationally. In Germany, for example, around 14,500 cases of PJI in hip and knee replacements occur annually. 5,100 of those are caused by multidrug resistant pathogens. ‘Eighty-seven percent of those affected die with- in five years,’ orthopaedic surgeon Professor Rudolf Ascherl MD pointed out during the Heraeus Symposium held at the German Congress of Orthopaedics and Traumatology (DKOU) held in Berlin, this October. Life expectancy is lower than for those affected by prostate cancer, breast cancer and melanoma, and quality of life is severely impaired. ‘The condition has chronic, almost oncological characteristics and patients suffer both physically and mentally for years,’ he added. ‘Twelve in 100 patients even state that they would rather die. We need a better understanding of who the high-risk patients actually are to get a better handle on PJI’, Ascherl believes. According to British studies, over- weight, multimorbid patients with heart, kidney or liver problems, and often further implants along with knee or hip endoprostheses, are at particular risk. ‘The present classification of PJI The experts gathered at the Heraeus Medical Satellite Symposium. From left: Professors Andrej Trampuz, Rudolf Ascherl, Klaus-Dieter Kühn and Volker Alt into acute or chronic stages does not sufficiently reflect the complex individual situation,’ according to Professor Volker Alt, from the Clinic and Polyclinic for Trauma Surgery at Regensburg University Hospital. A new classification of PJI devel- oped by Volker Alt and team now offers the prospect of improvement. This is to provide a clearer indica- tion if a patient is still suitable for an intervention, from which type of intervention they could benefit and if, like many of those affected, they have already undergone one or several revisions. ‘Like other clas- sification systems, PJI classification must consider all key factors, such as implant, pathogen and morbidi- ties. The condition of the soft tissue, concomitant diseases and whether or not the patient can be anaesthe- tised are also important. ‘The new classification may sound complicated,’ Alt observed, ‘but it’s basically designed to clarify every- thing that needs to be addressed in a structured manner, before any kind of treatment. ‘In future, all treatment options are to be based on this,’ Ascherl said. PJI is one of the main reasons for around 72,000 revisions of hip and knee joint replacements. These often complex interventions carry a risk of their own. Amputation is not uncommon. ‘If patients wear unsuitable spacers for too long, for instance, or if foreign bodies are not completely removed, we should talk about the option of amputation,’ Ascherl explained. He recommends this in cases where sep- sis and three relapses have already occurred. In each case, weighing up exarticulation and amputation must be on an individual basis. According to experts, the chal- lenge of PJI can only be met in a multidisciplinary, professional set- ting – with a consistently stand- ardised treatment algorithm, for instance – as implemented at the Centre for Musculoskeletal Surgery (CMSC) at the Charité University Hospital in Berlin. ‘Our multidis- ciplinary concept factors in spe- cifics such as nosocomial patho- gens, the condition of the bones and soft tissue, as well as the sta- bility of the endoprosthesis,’ said Professor Andrej Trampuz, Specialist in Infectious Diseases and Head of the Department of Septic Surgery at the Charité Berlin. The standardised procedure has reduced the occur- rence of relapses from 10.4% to 3.1%. ‘However, the increasing use of local antibiotics, implant coating and bacteriophages is new.’ Trampuz emphasised the impor- tance of treating PJI as early as possible. ‘If the implant is to be preserved, the infection must not be older than a maximum of four the weeks,’ infection specialist advised. During this acute phase, sufficient surgical debridement, the administration of systemic and local antibiotics and, if applicable, the exchange of the mobile prosthetic components, have priority. Once this early phase has passed, the persistent biofilms can no longer be eradicated even with biofilm- active antibiotics. ‘The objective is always the least invasive treatment option, with the best functional result without negative impact on the cure rate,’ Trampuz explained. ‘The importance of preserving the implant has increased considerably over recent years.’ After four weeks, PJI is regarded as chronic. There are around 6,500 of these late-stage infections every year, 4,000 in hip replacements and 2,500 in knee replacements. At this stage, those affected have now most definitely become high-risk patients. ‘If nosocomial pathogens are involved, when bones and soft tissue are in bad condition, and where the endoprosthesis is unsta- ble, the only options are complete revision or lifelong suppression,’ said Trampuz. The objective is always a one- stage exchange with a short inter- val. A Berlin study confirms the advantages. ‘Combined with local and systemic antibiotics this demon- strably reduces recurrences,’ he added. ‘During this late phase it’s important that the antibiotics are biofilm-active. Examples of this are rifampicin, ciprofloxacin, penicillin and fosfomycin. The combination of several anti- biotics in the bone cement is advo- cated to prevent resistance. The different active agents attack the bacterial metabolism in different EUROPEAN HOSPITAL Vol 28 Issue 6/19